Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2'-5' initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA. We find evidence for an expanded role of RNase H1 in the mechanism of RT-DNA termination, beyond the mere removal of RNA from RT-DNA:RNA hybrids. We show that endogenous RNase H1 determines the termination point of the retron RT-DNA, with differing effects across retron subtypes, and that these effects can be recapitulated using a reduced, in vitro system. We exclude mechanisms of termination that rely on steric effects of RNase H1 or RNA secondary structure and, instead, propose a model in which the tertiary structure of the single-stranded RT-DNA and remaining RNA template results in termination. Finally, we show that this mechanism affects cellular function, as retron-based phage defense is weaker in the absence of RNase H1.

Pyridostatin (PDS) is a well-known G-quadruplex (G4) inducer and stabilizer, yet its target genes have remained unclear. Herein, applying MS proteomics strategy, we revealed PDS significantly downregulated 22 proteins but upregulated 16 proteins in HeLa cancer cells, of which the genes both contain a number of G4 potential sequences, implying that PDS regulation on gene expression is far more complicated than inducing/stabilizing G4 structures. The PDS-downregulated proteins consequently upregulated 6 proteins to activate cyclin and cell cycle regulation, suggesting that PDS itself is not a potential anticancer agent, at least toward HeLa cancer cells. Importantly, SUB1, which encodes human positive cofactor and DNA lesion sensor PC4, was downregulated by 4.76-fold. Further studies demonstrated that the downregulation of PC4 dramatically promoted the cytotoxicity of trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) toward HeLa cells to a similar level of cisplatin, contributable to retarding the repair of 1,3-trans-PtTz crosslinked DNA lesion mediated by PC4. These findings not only provide new insights into better understanding on the biological functions of PDS but also implicate a strategy for the rational design of novel multi-targeting platinum anticancer drugs via conjugation of PDS as a ligand to the coordination scaffold of transplatin for battling drug resistance to cisplatin.

Alternative splicing is a key regulatory process underlying the amplification of genomic information and the expansion of proteomic diversity, particularly in brain. Here, we identify the Ewing sarcoma protein (EWS) as a new player of alternative splicing regulation during neuronal differentiation. Knockdown of EWS in neuronal progenitor cells leads to premature differentiation. Transcriptome profiling of EWS-depleted cells revealed global changes in splicing regulation. Bioinformatic analyses and biochemical experiments demonstrated that EWS regulates alternative exons in a position-dependent fashion. Notably, several EWS-regulated splicing events are physiologically modulated during neuronal differentiation and EWS depletion in neuronal precursors anticipates the splicing-pattern of mature neurons. Among other targets, we found that EWS controls the alternative splicing of the forkhead family transcription factor FOXP1, a pivotal transcriptional regulator of neuronal differentiation, possibly contributing to the switch of gene expression underlying the neuronal differentiation program.

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a major product of the DNA oxidization process, has been proposed to have an epigenetic function in gene regulation and has been associated with genome instability. NGS-based methodologies are contributing to the characterization of the 8-oxodG function in the genome. However, the 8-oxodG epigenetic role at a genomic level and the mechanisms controlling the genomic 8-oxodG accumulation/maintenance have not yet been fully characterized. In this study, we report the identification and characterization of a set of enhancer regions accumulating 8-oxodG in human epithelial cells. We found that these oxidized enhancers are mainly super-enhancers and are associated with bidirectional-transcribed enhancer RNAs and DNA Damage Response activation. Moreover, using ChIA-PET and HiC data, we identified specific CTCF-mediated chromatin loops in which the oxidized enhancer and promoter regions physically associate. Oxidized enhancers and their associated chromatin loops accumulate endogenous double-strand breaks which are in turn repaired by NHEJ pathway through a transcription-dependent mechanism. Our work suggests that 8-oxodG accumulation in enhancers-promoters pairs occurs in a transcription-dependent manner and provides novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers-promoters interactions.

Super enhancers (SEs) are broad enhancer domains usually containing multiple constituent enhancers that hold elevated activities in gene regulation. Disruption in one or more constituent enhancers causes aberrant SE activities that lead to gene dysregulation in diseases. To quantify SE aberrations, differential analysis is performed to compare SE activities between cell conditions. The state-of-art strategy in estimating differential SEs relies on overall activities and neglect the changes in length and structure of SEs. Here, we propose a novel computational method to identify differential SEs by weighting the combinatorial effects of constituent-enhancer activities and locations (i.e. internal dynamics). In addition to overall activity changes, our method identified four novel classes of differential SEs with distinct enhancer structural alterations. We demonstrate that these structure alterations hold distinct regulatory impact, such as regulating different number of genes and modulating gene expression with different strengths, highlighting the differentiated regulatory roles of these unexplored SE features. When compared to the existing method, our method showed improved identification of differential SEs that were linked to better discernment of cell-type-specific SE activity and functional interpretation.

Unnatural base pairs (UBPs) which exhibit a selectivity against pairing with canonical nucleobases provide a powerful tool for the development of nucleic acid-based technologies. As an alternative strategy to the conventional UBP designs, which involve utility of different recognition modes at the Watson-Crick interface, we now report that the exclusive base pairing can be achieved through the spatial separation of recognition units. The design concept was demonstrated with the alkynylated purine (NPu, OPu) and pyridazine (NPz, OPz) nucleosides endowed with nucleobase-like 2-aminopyrimidine or 2-pyridone ('pseudo-nucleobases') on their major groove side. These alkynylated purines and pyridazines exhibited exclusive and stable pairing properties by the formation of complementary hydrogen bonds between the pseudo-nucleobases in the DNA major groove as revealed by comprehensive Tm measurements, 2D-NMR analyses, and MD simulations. Moreover, the alkynylated purine-pyridazine pairs enabled dramatic stabilization of the DNA duplex upon consecutive incorporation while maintaining a high sequence-specificity. The present study showcases the separation of the recognition interface as a promising strategy for developing new types of UBPs.

Comment in Trends Plant Sci. 2022 Jul;27(7):624-626.

Genome instability has been recognized as a key driver for microbial and cancer adaptation and thus plays a central role in many diseases. Genome instability encompasses different types of genomic alterations, yet most available genome analysis software are limited to just one type of mutation. To overcome this limitation and better understand the role of genetic changes in enhancing pathogenicity we established GIP, a novel, powerful bioinformatic pipeline for comparative genome analysis. Here, we show its application to whole genome sequencing datasets of Leishmania, Plasmodium, Candida and cancer. Applying GIP on available data sets validated our pipeline and demonstrated the power of our tool to drive biological discovery. Applied to Plasmodium vivax genomes, our pipeline uncovered the convergent amplification of erythrocyte binding proteins and identified a nullisomic strain. Re-analyzing genomes of drug adapted Candida albicans strains revealed correlated copy number variations of functionally related genes, strongly supporting a mechanism of epistatic adaptation through interacting gene-dosage changes. Our results illustrate how GIP can be used for the identification of aneuploidy, gene copy number variations, changes in nucleic acid sequences, and chromosomal rearrangements. Altogether, GIP can shed light on the genetic bases of cell adaptation and drive disease biomarker discovery.

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. Hi-C is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any Hi-C experimental design. Here, we present the mathematical concepts to estimate the resolution of a dataset and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to several Hi-C libraries.

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case-specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq, and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease, and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays and showed comparable performance to commercial hot-start enzymes.

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1-/-) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1-/-, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.

Erratum in Nucleic Acids Res. 2022 May 20;50(9):5400.

DNA mechanical properties play a critical role in every aspect of DNA-dependent biological processes. Recently a high throughput assay named loop-seq has been developed to quantify the intrinsic bendability of a massive number of DNA fragments simultaneously. Using the loop-seq data, we develop a software tool, DNAcycP, based on a deep-learning approach for intrinsic DNA cyclizability prediction. We demonstrate DNAcycP predicts intrinsic DNA cyclizability with high fidelity compared to the experimental data. Using an independent dataset from in vitro selection for enrichment of loopable sequences, we further verified the predicted cyclizability score, termed C-score, can well distinguish DNA fragments with different loopability. We applied DNAcycP to multiple species and compared the C-scores with available high-resolution chemical nucleosome maps. Our analyses showed that both yeast and mouse genomes share a conserved feature of high DNA bendability spanning nucleosome dyads. Additionally, we extended our analysis to transcription factor binding sites and surprisingly found that the cyclizability is substantially elevated at CTCF binding sites in the mouse genome. We further demonstrate this distinct mechanical property is conserved across mammalian species and is inherent to CTCF binding DNA motif.

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes.

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.

Precision epigenome editing has gained significant attention as a method to modulate gene expression without altering genetic information. However, a major limiting factor has been that the gene expression changes are often transient, unlike the life-long epigenetic changes that occur frequently in nature. Here, we systematically interrogate the ability of CRISPR/dCas9-based epigenome editors (Epi-dCas9) to engineer persistent epigenetic silencing. We elucidated cis regulatory features that contribute to the differential stability of epigenetic reprogramming, such as the active transcription histone marks H3K36me3 and H3K27ac strongly correlating with resistance to short-term repression and resistance to long-term silencing, respectively. H3K27ac inversely correlates with increased DNA methylation. Interestingly, the dependance on H3K27ac was only observed when a combination of KRAB-dCas9 and targetable DNA methyltransferases (DNMT3A-dCas9 + DNMT3L) was used, but not when KRAB was replaced with the targetable H3K27 histone methyltransferase Ezh2. In addition, programmable Ezh2/DNMT3A + L treatment demonstrated enhanced engineering of localized DNA methylation and was not sensitive to a divergent chromatin state. Our results highlight the importance of local chromatin features for heritability of programmable silencing and the differential response to KRAB- and Ezh2-based epigenetic editing platforms. The information gained in this study provides fundamental insights into understanding contextual cues to more predictably engineer persistent silencing.

Bovine leukemia virus (BLV)-induced tumoral development is a multifactorial phenomenon that remains incompletely understood. Here, we highlight the critical role of the cellular CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the three-dimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the provirus. Next, we showed that CTCF localized to regions of transitions in the histone modifications profile along the BLV genome and that it is implicated in the repression of the 5'Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3'LTR promoter activity. Finally, we demonstrated that BLV integration deregulated the host cellular 3D chromatin organization through the formation of viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.

Identifying rare variants that contribute to complex diseases is challenging because of the low statistical power in current tests comparing cases with controls. Here, we propose a novel and powerful rare variants association test based on the deviation of the observed mutation burden of a gene in cases from a baseline predicted by a weighted recursive truncated negative-binomial regression (RUNNER) on genomic features available from public data. Simulation studies show that RUNNER is substantially more powerful than state-of-the-art rare variant association tests and has reasonable type 1 error rates even for stratified populations or in small samples. Applied to real case-control data, RUNNER recapitulates known genes of Hirschsprung disease and Alzheimer's disease missed by current methods and detects promising new candidate genes for both disorders. In a case-only study, RUNNER successfully detected a known causal gene of amyotrophic lateral sclerosis. The present study provides a powerful and robust method to identify susceptibility genes with rare risk variants for complex diseases.

Despite a vast expansion in the availability of epigenomic data, our knowledge of the chromatin landscape at interspersed repeats remains highly limited by difficulties in mapping short-read sequencing data to these regions. In particular, little is known about the locus-specific regulation of evolutionarily young transposable elements (TEs), which have been implicated in genome stability, gene regulation and innate immunity in a variety of developmental and disease contexts. Here we propose an approach for generating locus-specific protein-DNA binding profiles at interspersed repeats, which leverages information on the spatial proximity between repetitive and non-repetitive genomic regions. We demonstrate that the combination of HiChIP and a newly developed mapping tool (PAtChER) yields accurate protein enrichment profiles at individual repetitive loci. Using this approach, we reveal previously unappreciated variation in the epigenetic profiles of young TE loci in mouse and human cells. Insights gained using our method will be invaluable for dissecting the molecular determinants of TE regulation and their impact on the genome.

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.

Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

Oligonucleotide (ON) therapeutics are emerging as a new generation of medicine with tremendous potential, but their clinical translation is hampered by inferior stability and short circulation time in the human body. Here, we report a general approach to manipulating the interaction between ONs and albumin by modulating hydrophobicity. A series of DNA aptamer derivatives were designed and prepared by programmable synthesis as an ON library with a gradient of hydrophobic base 'F'. In vitro experiments revealed that the introduction of two F bases at both ends of ONs enhanced the biostability without sacrificing biological activities, while the binding affinity toward albumin was dramatically increased with Kd in the range of 100 nM to 1 μM. In vivo imaging confirmed the immediate formation of the aptamer-albumin complex after the injection, and the circulation time of the aptamer was dramatically elongated owing to the enhanced biostability and retarded renal excretion. The programmable incorporation of the F base provides a general approach to regulating albumin-binding affinity and enhancing the stability of aptamers in vivo, conferring aptamer therapeutics prolonged circulation time to meet clinical requirements.

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.

Aging in humans is intricately linked with alterations in circadian rhythms concomitant with physiological decline and stem cell exhaustion. However, whether the circadian machinery directly regulates stem cell aging, especially in primates, remains poorly understood. In this study, we found that deficiency of BMAL1, the only non-redundant circadian clock component, results in an accelerated aging phenotype in both human and cynomolgus monkey mesenchymal progenitor cells (MPCs). Unexpectedly, this phenotype was mainly attributed to a transcription-independent role of BMAL1 in stabilizing heterochromatin and thus preventing activation of the LINE1-cGAS-STING pathway. In senescent primate MPCs, we observed decreased capacity of BMAL1 to bind to LINE1 and synergistic activation of LINE1 expression. Likewise, in the skin and muscle tissues from the BMAL1-deficient cynomolgus monkey, we observed destabilized heterochromatin and aberrant LINE1 transcription. Altogether, these findings uncovered a noncanonical role of BMAL1 in stabilizing heterochromatin to inactivate LINE1 that drives aging in primate cells.

Concatemers of d(TCCC) that were first detected through their association with deletions at the RACK7 locus, are widespread throughout the human genome. Circular dichroism spectra show that d(GGGA)n sequences form G-quadruplexes when n > 3, while i-motif structures form at d(TCCC)n sequences at neutral pH when n ≥ 7 in vitro. In the PC3 cell line, deletions are observed only when the d(TCCC)n variant is long enough to form significant levels of unresolved i-motif structure at neutral pH. The presence of an unresolved i-motif at a representative d(TCCC)n element at RACK7 was suggested by experiments showing that that the region containing the d(TCCC)9 element was susceptible to bisulfite attack in native DNA and that d(TCCC)9 oligo formed an i-motif structure at neutral pH. This in turn suggested that that the i-motif present at this site in native DNA must be susceptible to bisulfite mediated deamination even though it is a closed structure. Bisulfite deamination of the i-motif structure in the model oligodeoxynucleotide was confirmed using mass spectrometry analysis. We conclude that while G-quadruplex formation may contribute to spontaneous mutation at these sites, deletions actually require the potential for i-motif to form and remain unresolved at neutral pH.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA-ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.

Cellular DNA is continuously transcribed into RNA by multisubunit RNA polymerases (RNAPs). The continuity of transcription can be disrupted by DNA lesions that arise from the activities of cellular enzymes, reactions with endogenous and exogenous chemicals or irradiation. Here, we review available data on translesion RNA synthesis by multisubunit RNAPs from various domains of life, define common principles and variations in DNA damage sensing by RNAP, and consider existing controversies in the field of translesion transcription. Depending on the type of DNA lesion, it may be correctly bypassed by RNAP, or lead to transcriptional mutagenesis, or result in transcription stalling. Various lesions can affect the loading of the templating base into the active site of RNAP, or interfere with nucleotide binding and incorporation into RNA, or impair RNAP translocation. Stalled RNAP acts as a sensor of DNA damage during transcription-coupled repair. The outcome of DNA lesion recognition by RNAP depends on the interplay between multiple transcription and repair factors, which can stimulate RNAP bypass or increase RNAP stalling, and plays the central role in maintaining the DNA integrity. Unveiling the mechanisms of translesion transcription in various systems is thus instrumental for understanding molecular pathways underlying gene regulation and genome stability.

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.

Heterochromatin-associated gene silencing controls multiple physiological processes in malaria parasites, however, little is known concerning the regulatory network and cis-acting sequences involved in the organization of heterochromatin and how they modulate heterochromatic gene expression. Based on systematic profiling of genome-wide occupancy of eighteen Apicomplexan AP2 transcription factors by ChIP-seq analysis, we identify and characterize eight heterochromatin-associated factors (PfAP2-HFs), which exhibit preferential enrichment within heterochromatic regions but with differential coverage profiles. Although these ApiAP2s target euchromatic gene loci via specific DNA motifs, they are likely integral components of heterochromatin independent of DNA motif recognition. Systematic knockout screenings of ApiAP2 factors coupled with RNA-seq transcriptomic profiling revealed three activators and three repressors of heterochromatic gene expression including four PfAP2-HFs. Notably, expression of virulence genes is either completely silenced or significantly reduced upon the depletion of PfAP2-HC. Integrated multi-omics analyses reveal autoregulation and feed-forward loops to be common features of the ApiAP2 regulatory network, in addition to the occurrence of dynamic interplay between local chromatin structure and ApiAP2s in transcriptional control. Collectively, this study provides a valuable resource describing the genome-wide landscape of the ApiAP2 family and insights into functional divergence and cooperation within this family during the blood-stage development of malaria parasites.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction-modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive model, called Exon ByPASS (predicting Exon skipping Based on Protein amino acid SequenceS), to assess the criticality of exon inclusion based solely on information contained in the amino acid sequence upstream and downstream of the exon junctions. By focusing on protein sequence, Exon ByPASS predicts exon skipping independent of tissue and species in the absence of any intronic information. We validate model predictions using transcriptomic and proteomic data and show that the model can capture exon skipping in different tissues and species. Additionally, we reveal potential therapeutic opportunities by predicting synthetically skippable exons and neo-junctions arising in cancer cells.

CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window. The impact of features on editing rate depends on the position, with sequence bias efficacy mainly influencing bases away from the center of the window. We use these observations to train a machine learning model to predict editing activity per position, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of disease mutation correcting guides, and find that most of them suffer from more unwanted editing than pure outcomes. This work unravels the position-specificity of base editing biases and allows more efficient planning of editing campaigns in experimental and therapeutic contexts.

CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

The 48 human nuclear receptors (NRs) form a superfamily of transcription factors that regulate major physiological and pathological processes. Emerging evidence suggests that NR crosstalk can fundamentally change our understanding of NR biology, but detailed molecular mechanisms of crosstalk are lacking. Here, we report the molecular basis of crosstalk between the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), where they form a novel heterodimer, resulting in their mutual inhibition. PXR and CAR regulate drug metabolism and energy metabolism. Although they have been broadly perceived as functionally redundant, a growing number of reports suggests a mutual inhibitory relation, but their precise mode of coordinated action remains unknown. Using methods including RNA sequencing, small-angle X-ray scattering and crosslinking mass spectrometry we demonstrate that the mutual inhibition altered gene expression globally and is attributed to the novel PXR-CAR heterodimerization via the same interface used by each receptor to heterodimerize with its functional partner, retinoid X receptor (RXR). These findings establish an unexpected functional relation between PXR, CAR and RXR, change the perceived functional relation between PXR and CAR, open new perspectives on elucidating their role and designing approaches to regulate them, and highlight the importance to comprehensively investigate nuclear receptor crosstalk.

Detection of low-level DNA mutations can reveal recurrent, hotspot genetic changes of clinical relevance to cancer, prenatal diagnostics, organ transplantation or infectious diseases. However, the high excess of wild-type (WT) alleles, which are concurrently present, often hinders identification of salient genetic changes. Here, we introduce UV-mediated cross-linking minor allele enrichment (UVME), a novel approach that incorporates ultraviolet irradiation (∼365 nm UV) DNA cross-linking either before or during PCR amplification. Oligonucleotide probes matching the WT target sequence and incorporating a UV-sensitive 3-cyanovinylcarbazole nucleoside modification are employed for cross-linking WT DNA. Mismatches formed with mutated alleles reduce DNA binding and UV-mediated cross-linking and favor mutated DNA amplification. UV can be applied before PCR and/or at any stage during PCR to selectively block WT DNA amplification and enable identification of traces of mutated alleles. This enables a single-tube PCR reaction directly from genomic DNA combining optimal pre-amplification of mutated alleles, which then switches to UV-mediated mutation enrichment-based DNA target amplification. UVME cross-linking enables enrichment of mutated KRAS and p53 alleles, which can be screened directly via Sanger sequencing, high-resolution melting, TaqMan genotyping or digital PCR, resulting in the detection of mutation allelic frequencies of 0.001-0.1% depending on the endpoint detection method. UV-mediated mutation enrichment provides new potential for mutation enrichment in diverse clinical samples.

Despite recent methodology and reference database improvements for taxonomic profiling tools, metagenomic assembly and genomic binning remain important pillars of metagenomic analysis workflows. In case reference information is lacking, genomic binning is considered to be a state-of-the-art method in mixed culture metagenomic data analysis. In this light, our previously published tool BusyBee Web implements a composition-based binning method efficient enough to function as a rapid online utility. Handling assembled contigs and long nanopore generated reads alike, the webserver provides a wide range of supplementary annotations and visualizations. Half a decade after the initial publication, we revisited existing functionality, added comprehensive visualizations, and increased the number of data analysis customization options for further experimentation. The webserver now allows for visualization-supported differential analysis of samples, which is computationally expensive and typically only performed in coverage-based binning methods. Further, users may now optionally check their uploaded samples for plasmid sequences using PLSDB as a reference database. Lastly, a new application programming interface with a supporting python package was implemented, to allow power users fully automated access to the resource and integration into existing workflows. The webserver is freely available under: https://www.ccb.uni-saarland.de/busybee.

Cellular regulation is inherently complex, and one particular cellular function is often controlled by a cascade of different types of regulatory interactions. For example, the activity of a transcription factor (TF), which regulates the expression level of downstream genes through transcriptional regulation, can be regulated by small molecules through compound-protein interactions. To identify such complex regulatory cascades, traditional relational databases require ineffective additional operations and are computationally expensive. In contrast, graph databases are purposefully developed to execute such deep searches efficiently. Here, we present ERMer (E. coli Regulation Miner), the first cloud platform for mining the regulatory landscape of Escherichia coli based on graph databases. Combining the AWS Neptune graph database, AWS lambda function, and G6 graph visualization engine enables quick search and visualization of complex regulatory cascades/patterns. Users can also interactively navigate the E. coli regulatory landscape through ERMer. Furthermore, a Q&A module is included to showcase the power of graph databases in answering complex biological questions through simple queries. The backend graph model can be easily extended as new data become available. In addition, the framework implemented in ERMer can be easily migrated to other applications or organisms. ERMer is available at https://ermer.biodesign.ac.cn/.

The prediction of protein subcellular localization is of great relevance for proteomics research. Here, we propose an update to the popular tool DeepLoc with multi-localization prediction and improvements in both performance and interpretability. For training and validation, we curate eukaryotic and human multi-location protein datasets with stringent homology partitioning and enriched with sorting signal information compiled from the literature. We achieve state-of-the-art performance in DeepLoc 2.0 by using a pre-trained protein language model. It has the further advantage that it uses sequence input rather than relying on slower protein profiles. We provide two means of better interpretability: an attention output along the sequence and highly accurate prediction of nine different types of protein sorting signals. We find that the attention output correlates well with the position of sorting signals. The webserver is available at services.healthtech.dtu.dk/service.php?DeepLoc-2.0.

With the shift from SNP arrays to high-throughput sequencing, most researchers studying diseases in consanguineous families do not rely on linkage analysis any longer, but simply search for deleterious variants which are homozygous in all patients. AutozygosityMapper allows the fast and convenient identification of disease mutations in patients from consanguineous pedigrees by focussing on homozygous segments shared by all patients. Users can upload multi-sample VCF files, including WGS data, without any pre-processing. Genome-wide runs of homozygosity and the underlying genotypes are presented in graphical interfaces. AutozygosityMapper extends the functions of its predecessor, HomozygosityMapper, to the search for autozygous regions, in which all patients share the same homozygous genotype. We provide export of VCF files containing only the variants found in homozygous regions, this usually reduces the number of variants by two orders of magnitude. These regions can also directly be analysed with our disease mutation identification tool MutationDistiller. The application comes with simple and intuitive graphical interfaces for data upload, analysis, and results. We kept the structure of HomozygosityMapper so that previous users will find it easy to switch. With AutozygosityMapper, we provide a fast web-based way to identify disease mutations in consanguineous families. AutozygosityMapper is freely available at https://www.genecascade.org/AutozygosityMapper/.

Upon the ever-increasing number of publicly available experimentally determined and predicted protein and nucleic acid structures, the demand for easy-to-use tools to investigate these structural models is higher than ever before. The ProteinsPlus web server (https://proteins.plus) comprises a growing collection of molecular modeling tools focusing on protein-ligand interactions. It enables quick access to structural investigations ranging from structure analytics and search methods to molecular docking. It is by now well-established in the community and constantly extended. The server gives easy access not only to experts but also to students and occasional users from the field of life sciences. Here, we describe its recently added new features and tools, beyond them a novel method for on-the-fly molecular docking and a search method for single-residue substitutions in local regions of a protein structure throughout the whole Protein Data Bank. Finally, we provide a glimpse into new avenues for the annotation of AlphaFold structures which are directly accessible via a RESTful service on the ProteinsPlus web server.

Establishing the relationship between protein-coding genes and phenotypes has the potential to inform on the molecular etiology of diseases. Here, we describe ExPheWas (exphewas.ca), a gene-based phenome-wide association study browser and platform that enables the conduct of gene-based Mendelian randomization. The ExPheWas data repository includes sex-stratified and sex-combined gene-based association results from 26 616 genes with 1746 phenotypes measured in up to 413 133 individuals from the UK Biobank. Interactive visualizations are provided through a browser to facilitate data exploration supported by false discovery rate control, and it includes tools for enrichment analysis. The interactive Mendelian randomization module in ExPheWas allows the estimation of causal effects of a genetically predicted exposure on an outcome by using genetic variation in a single gene as the instrumental variable.

Phase separation-based condensate formation is a novel working paradigm in biology, helping to rationalize many important cellular phenomena including the assembly of membraneless organelles. Uncovering the functional impact of cellular condensates requires a better knowledge of these condensates' constituents. Herein, we introduce the webserver GraPES (Granule Protein Enrichment Server), a user-friendly online interface containing the MaGS and MaGSeq predictors, which provide propensity scores for proteins' localization into cellular condensates. Our webpage contains models trained on human (Homo sapiens) and yeast (Saccharomyces cerevisiae) stress granule proteins. MaGS utilizes experimentally-based protein features for prediction, whereas MaGSeq is an entirely protein sequence-based implementation. GraPES is implemented in HTML/CSS and Javascript and is freely available for public use at https://grapes.msl.ubc.ca/. Documentation for using the provided webtools, descriptions of their methodology, and implementation notes can be found on the webpage.

K-mers are short DNA sequences that are used for genome sequence analysis. Applications that use k-mers include genome assembly and alignment. However, the wider bioinformatic use of these short sequences has challenges related to the massive scale of genomic sequence data. A single human genome assembly has billions of k-mers. As a result, the computational requirements for analyzing k-mer information is enormous, particularly when involving complete genome assemblies. To address these issues, we developed a new indexing data structure based on a hash table tuned for the lookup of short sequence keys. This web application, referred to as KmerKeys, provides performant, rapid query speeds for cloud computation on genome assemblies. We enable fuzzy as well as exact sequence searches of assemblies. To enable robust and speedy performance, the website implements cache-friendly hash tables, memory mapping and massive parallel processing. Our method employs a scalable and efficient data structure that can be used to jointly index and search a large collection of human genome assembly information. One can include variant databases and their associated metadata such as the gnomAD population variant catalogue. This feature enables the incorporation of future genomic information into sequencing analysis. KmerKeys is freely accessible at https://kmerkeys.dgi-stanford.org.

Periodic gene expression dynamics are key to cell and organism physiology. Studies of oscillatory expression have focused on networks with intuitive regulatory negative feedback loops, leaving unknown whether other common biochemical reactions can produce oscillations. Oscillation and noise have been proposed to support mammalian progenitor cells' capacity to restore heterogenous, multimodal expression from extreme subpopulations, but underlying networks and specific roles of noise remained elusive. We use mass-action-based models to show that regulated RNA degradation involving as few as two RNA species-applicable to nearly half of human protein-coding genes-can generate sustained oscillations without explicit feedback. Diverging oscillation periods synergize with noise to robustly restore cell populations' bimodal expression on timescales of days. The global bifurcation organizing this divergence relies on an oscillator and bistable switch which cannot be decomposed into two structural modules. Our work reveals surprisingly rich dynamics of post-transcriptional reactions and a potentially widespread mechanism underlying development, tissue regeneration, and cancer cell heterogeneity.

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 Å in other phages, spacings of 33-36 Å between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three 'AC(A/G)AGG'-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236-286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.

The nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small, excised damage-containing DNA oligonucleotides (sedDNAs) ∼30 nt in length. How cells process and degrade these byproducts of DNA repair is not known. Using a small scale RNA interference screen in UV-irradiated human cells, we identified TREX1 as a major regulator of sedDNA abundance. Knockdown of TREX1 increased the level of sedDNAs containing the two major UV photoproducts and their association with the NER proteins TFIIH and RPA. Overexpression of wild-type but not nuclease-inactive TREX1 significantly diminished sedDNA levels, and studies with purified recombinant TREX1 showed that the enzyme efficiently degrades DNA located 3' of the UV photoproduct in the sedDNA. Knockdown or overexpression of TREX1 did not impact the overall rate of UV photoproduct removal from genomic DNA or cell survival, which indicates that TREX1 function in sedDNA degradation does not impact NER efficiency. Taken together, these results indicate a previously unknown role for TREX1 in promoting the degradation of the sedDNA products of the repair reaction. Because TREX1 mutations and inefficient DNA degradation impact inflammatory and immune signaling pathways, the regulation of sedDNA degradation by TREX1 may contribute to photosensitive skin disorders.

Reactive oxygen species (ROS) are constantly produced in cells, an excess of which causes oxidative stress. ROS has been linked to regulation of the Hippo pathway; however, the underlying detailed mechanisms remain unclear. Here, we report that MOB1, a substrate of MST1/2 and co-activator of LATS1/2 in the canonical Hippo pathway, interacts with and is acetylated at lysine 11 by acetyltransferase CBP and deacetylated by HDAC6. MOB1-K11 acetylation stabilizes itself by reducing its binding capacity with E3 ligase Praja2 and subsequent ubiquitination. MOB1-K11 acetylation increases its phosphorylation and activates LATS1. Importantly, upstream oxidative stress signals promote MOB1 acetylation by suppressing CBP degradation, independent of MST1/2 kinase activity and HDAC6 deacetylation effect, thereby linking oxidative stress to activation of the Hippo pathway. Functionally, the acetylation-deficient mutant MOB1-K11R promotes lung cancer cell proliferation, migration and invasion in vitro and accelerates tumor growth in vivo, compared to the wild-type MOB1. Clinically, acetylated MOB1 corresponds to better prediction of overall survival in patients with non-small cell lung cancer. Therefore, as demonstrated, an oxidative stress-CBP regulatory axis controls MOB1-K11 acetylation and activates LATS1, thereby activating the Hippo pathway and suppressing YAP/TAZ nuclear translocation and tumor progression.

During the cellular adaptation to nutrient starvation, cells temporarily decelerate translation processes including ribosomal biogenesis. However, the mechanisms repressing robust gene expression from the ribosomal gene cluster (rDNA) are unclear. Here, we demonstrate that fission yeast cells facing glucose starvation assemble facultative heterochromatin in rDNA leading to its transcriptional repression. Glucose starvation induces quick dissociation of the ATF/CREB-family protein Atf1 from rDNA, where in turn the histone chaperone FACT is recruited to promote H3K9 methylation and heterochromatinization. We also identify the histone acetyltransferase Gcn5 as a repressor of rDNA heterochromatinization in glucose-rich conditions, and this protein dissociates from rDNA upon glucose starvation. Facultative heterochromatin formation in rDNA requires histone deacetylases Clr3 and both the RNAi-dependent and -independent gene silencing pathways. This is essential in adaptation to starvation since mutants lacking heterochromatin formation in rDNA lead to untimely cell death during glucose starvation.

Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain upstream of the cat-3 gene, hereby called 5H-cat-3 domain, to constrain aberrant heterochromatin spreading. Interestingly, aberrant spreading of the 5H-cat-3 domain observed in the dmm-1KO strain is accompanied by robust deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5H-cat-3 domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5H-cat-3 domain. Importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin. Together, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson-Crick base pair at the second position of the anticodon-codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

The core catalytic unit of telomerase comprises telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). Unlike TERT, which is predominantly expressed in cancer and stem cells, TERC is ubiquitously expressed in normal somatic cells without telomerase activity. However, the functions of TERC in these telomerase-negative cells remain elusive. Here, we reported positive feedback regulation between TERC and the PI3K-AKT pathway that controlled cell proliferation independent of telomerase activity in human fibroblasts. Mechanistically, we revealed that TERC activated the transcription of target genes from the PI3K-AKT pathway, such as PDPK1, by targeting their promoters. Overexpression of PDPK1 partially rescued the deficiency of AKT activation caused by TERC depletion. Furthermore, we found that FOXO1, a transcription factor negatively regulated by the PI3K-AKT pathway, bound to TERC promoter and suppressed its expression. Intriguingly, TERC-induced activation of the PI3K-AKT pathway also played a critical role in the proliferation of activated CD4+ T cells. Collectively, our findings identify a novel function of TERC that regulates the PI3K-AKT pathway via positive feedback to elevate cell proliferation independent of telomerase activity and provide a potential strategy to promote CD4+ T cells expansion that is responsible for enhancing adaptive immune reactions to defend against pathogens and tumor cells.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Adaptation of viruses to their environments occurs through the acquisition of both novel single-nucleotide variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a 'one-to-more' ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.

Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN-MCM-GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD's DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N-Gins51C-GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa coordinates cellular processes in response to stimuli. We used 364 transcriptomes (281 publicly available + 83 in-house generated) to reconstruct the TRN of P. aeruginosa using independent component analysis. We identified 104 independently modulated sets of genes (iModulons) among which 81 reflect the effects of known transcriptional regulators. We identified iModulons that (i) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs), (ii) show increased expression of the BGCs and associated secretion systems in nutrient conditions that are important in cystic fibrosis, (iii) show the presence of a novel ribosomally synthesized and post-translationally modified peptide (RiPP) BGC which might have a role in P. aeruginosa virulence, (iv) exhibit interplay of amino acid metabolism regulation and central metabolism across different carbon sources and (v) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compared the identified iModulons of P. aeruginosa with those previously described in Escherichia coli to observe conserved regulons across two Gram-negative species. This comprehensive TRN framework encompasses the majority of the transcriptional regulatory machinery in P. aeruginosa, and thus should prove foundational for future research into its physiological functions.

Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson-Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m3C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m3C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m3C32 modification of human mitochondrial tRNAThr (hmtRNAThr) was not reliant on t6A modification at A37 (t6A37), while that of hmtRNASer(UCN) critically depended on i6A modification at A37 (i6A37). We clarified the hmtRNAThr substrate recognition mechanism, which was obviously different from that of hmtRNASer(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m3C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m3C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

Erratum in Nucleic Acids Res. 2022 Apr 07;:

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

Proteins encoded by newly-emerged genes ('orphan genes') share no sequence similarity with proteins in any other species. They provide organisms with a reservoir of genetic elements to quickly respond to changing selection pressures. Here, we systematically assess the ability of five gene prediction pipelines to accurately predict genes in genomes according to phylostratal origin. BRAKER and MAKER are existing, popular ab initio tools that infer gene structures by machine learning. Direct Inference is an evidence-based pipeline we developed to predict gene structures from alignments of RNA-Seq data. The BIND pipeline integrates ab initio predictions of BRAKER and Direct inference; MIND combines Direct Inference and MAKER predictions. We use highly-curated Arabidopsis and yeast annotations as gold-standard benchmarks, and cross-validate in rice. Each pipeline under-predicts orphan genes (as few as 11 percent, under one prediction scenario). Increasing RNA-Seq diversity greatly improves prediction efficacy. The combined methods (BIND and MIND) yield best predictions overall, BIND identifying 68% of annotated orphan genes, 99% of ancient genes, and give the highest sensitivity score regardless dataset in Arabidopsis. We provide a light weight, flexible, reproducible, and well-documented solution to improve gene prediction.

Methods that can randomly introduce mutations in the microbial genome have been used for classical genetic screening and, more recently, the evolutionary engineering of microbial cells. However, most methods rely on either cell-damaging agents or disruptive mutations of genes that are involved in accurate DNA replication, of which the latter requires prior knowledge of gene functions, and thus, is not easily transferable to other species. In this study, we developed a new mutator for in vivo mutagenesis that can directly modify the genomic DNA. Mutator protein, MutaEco, in which a DNA-modifying enzyme is fused to the α-subunit of Escherichia coli RNA polymerase, increases the mutation rate without compromising the cell viability and accelerates the adaptive evolution of E. coli for stress tolerance and utilization of unconventional carbon sources. This fusion strategy is expected to accommodate diverse DNA-modifying enzymes and may be easily adapted to various bacterial species.

Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level interaction of DNA is a foundational and powerful mechanism to build DNA structures at the nanoscale; however, its temperature sensitivity and weak interaction force remain a barrier for the facile and scalable assembly of DNA structures toward higher-order structures. We conducted this study to provide an alternative, non-base-pairing approach to connect nanoscale DNA units to yield micrometer-sized gels based on the sequential phase transition of amphiphilic unit structures. Strong electrostatic interactions between DNA nanostructures and polyelectrolyte spermines led to the formation of giant phase-separated aggregates of monomer units. Gelation could be initiated by the addition of NaCl, which weakened the electrostatic DNA-spermine interaction while attractive interactions between cholesterols created stable networks by crosslinking DNA monomers. In contrast to the conventional DNA gelation techniques, our system used solid aggregates as a precursor for DNA microgels. Therefore, in situ gelation could be achieved by depositing aggregates on the desired substrate and subsequently initiating a phase transition. Our approach can expand the utility and functionality of DNA hydrogels by using more complex nucleic acid assemblies as unit structures and combining the technique with top-down microfabrication methods.

As the design of genetic circuitry for synthetic biology becomes more sophisticated, diverse regulatory bioparts are required. Despite their importance, well-characterized 3'-untranslated region (3'-UTR) bioparts are limited. Thus, transcript 3'-ends require further investigation to understand the underlying regulatory role and applications of the 3'-UTR. Here, we revisited the use of Term-Seq in the Escherichia coli strain K-12 MG1655 to enhance our understanding of 3'-UTR regulatory functions and to provide a diverse collection of tunable 3'-UTR bioparts with a wide termination strength range. Comprehensive analysis of 1,629 transcript 3'-end positions revealed multiple 3'-termini classes generated through transcription termination and RNA processing. The examination of individual Rho-independent terminators revealed a reduction in downstream gene expression over a wide range, which led to the design of novel synthetic metabolic valves that control metabolic fluxes in branched pathways. These synthetic metabolic valves determine the optimal balance of heterologous pathways for maximum target biochemical productivity. The regulatory strategy using 3'-UTR bioparts is advantageous over promoter- or 5'-UTR-based transcriptional control as it modulates gene expression at transcription levels without trans-acting element requirements (e.g. transcription factors). Our results provide a foundational platform for 3'-UTR engineering in synthetic biology applications.

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double-stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3'-phospho-α,β-unsaturated aldehyde (3'-PUA), to form unstable but long-lived Schiff base DNA-protein cross-links at 3'-DNA termini (3'-PUA-protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1's intrinsic redox capacity, yielding a stable 3'-PUA-PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3'-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3'-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3'-PUA-protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3'-PUA-histone DPCs, but not 3'-PUA-PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3'-PUA-protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Dual-inhibitors of PARP1 and PARP2 are promising anti-cancer drugs. In addition to blocking PARP1&2 enzymatic activity, PARP inhibitors also extend the lifetime of DNA damage-induced PARP1&2 foci, termed trapping. Trapping is important for the therapeutic effects of PARP inhibitors. Using live-cell imaging, we found that PARP inhibitors cause persistent PARP2 foci by switching the mode of PARP2 recruitment from a predominantly PARP1- and PAR-dependent rapid exchange to a WGR domain-mediated stalling of PARP2 on DNA. Specifically, PARP1-deletion markedly reduces but does not abolish PARP2 foci. The residual PARP2 foci in PARP1-deficient cells are DNA-dependent and abrogated by the R140A mutation in the WGR domain. Yet, PARP2-R140A forms normal foci in PARP1-proficient cells. In PARP1-deficient cells, PARP inhibitors - niraparib, talazoparib, and, to a lesser extent, olaparib - enhance PARP2 foci by preventing PARP2 exchange. This trapping of PARP2 is independent of auto-PARylation and is abolished by the R140A mutation in the WGR domain and the H415A mutation in the catalytic domain. Taken together, we found that PARP inhibitors trap PARP2 by physically stalling PARP2 on DNA via the WGR-DNA interaction while suppressing the PARP1- and PAR-dependent rapid exchange of PARP2.

The human genome encodes large numbers of non-coding RNAs, including divergent antisense transcripts at transcription start sites (TSSs). However, molecular mechanisms by which divergent antisense transcription is regulated have not been detailed. Here, we report a novel ZWC complex composed of ZC3H4, WDR82 and CK2 that suppresses divergent antisense transcription. The ZWC complex preferentially localizes at TSSs of active genes through direct interactions of ZC3H4 and WDR82 subunits with the S5p RNAPII C-terminal domain. ZC3H4 depletion leads to increased divergent antisense transcription, especially at genes that naturally produce divergent antisense transcripts. We further demonstrate that the ZWC complex phosphorylates the previously uncharacterized N-terminal acidic domain of SPT5, a subunit of the transcription-elongation factor DSIF, and that this phosphorylation is responsible for suppressing divergent antisense transcription. Our study provides evidence that the newly identified ZWC-DSIF axis regulates the direction of transcription during the transition from early to productive elongation.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.

Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation.

Erratum in Nucleic Acids Res. 2022 Mar 29;:

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.

Haploinsufficiency drives Darwinian evolution. Siblings, while alike in many aspects, differ due to monoallelic differences inherited from each parent. In cancer, solid tumors exhibit aneuploid genetics resulting in hundreds to thousands of monoallelic gene-level copy-number alterations (CNAs) in each tumor. Aneuploidy patterns are heterogeneous, posing a challenge to identify drivers in this high-noise genetic environment. Here, we developed Shifted Weighted Annotation Network (SWAN) analysis to assess biology impacted by cumulative monoallelic changes. SWAN enables an integrated pathway-network analysis of CNAs, RNA expression, and mutations via a simple web platform. SWAN is optimized to best prioritize known and novel tumor suppressors and oncogenes, thereby identifying drivers and potential druggable vulnerabilities within cancer CNAs. Protein homeostasis, phospholipid dephosphorylation, and ion transport pathways are commonly suppressed. An atlas of CNA pathways altered in each cancer type is released. These CNA network shifts highlight new, attractive targets to exploit in solid tumors.

Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1.2/H1.4 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local GC content and that their distribution is robust with respect to H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of topologically associating domains (TADs). Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells.

Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA en route to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.

During packaging in positive-sense single-stranded RNA (+ssRNA) viruses, coat proteins (CPs) interact directly with multiple regions in genomic RNA (gRNA), but the underlying physicochemical principles remain unclear. Here we analyze the high-resolution cryo-EM structure of bacteriophage MS2 and show that the gRNA/CP binding sites, including the known packaging signal, overlap significantly with regions where gRNA nucleobase-density profiles match the corresponding CP nucleobase-affinity profiles. Moreover, we show that the MS2 packaging signal corresponds to the global minimum in gRNA/CP interaction energy in the unstructured state as derived using a linearly additive model and knowledge-based nucleobase/amino-acid affinities. Motivated by this, we predict gRNA/CP interaction sites for a comprehensive set of 1082 +ssRNA viruses. We validate our predictions by comparing them with site-resolved information on gRNA/CP interactions derived in SELEX and CLIP experiments for 10 different viruses. Finally, we show that in experimentally studied systems CPs frequently interact with autologous coding regions in gRNA, in agreement with both predicted interaction energies and a recent proposal that proteins in general tend to interact with own mRNAs, if unstructured. Our results define a self-consistent framework for understanding packaging in +ssRNA viruses and implicate interactions between unstructured gRNA and CPs in the process.

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR-Cas9 guide design.

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.

Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.

GWASs have identified numerous genetic variants associated with a wide variety of diseases, yet despite the wide availability of genetic testing the insights that would enhance the interpretability of these results are not widely available to members of the public. As a proof of concept and demonstration of technological feasibility, we developed PAGEANT (Personal Access to Genome & Analysis of Natural Traits), usable through Graphical User Interface or command line-based version, aiming to serve as a protocol and prototype that guides the overarching design of genetic reporting tools. PAGEANT is structured across five core modules, summarized by five Qs: (i) quality assurance of the genetic data; (ii) qualitative assessment of genetic characteristics; (iii) quantitative assessment of health risk susceptibility based on polygenic risk scores and population reference; (iv) query of third-party variant databases (e.g. ClinVAR and PharmGKB) and (v) quick Response code of genetic variants of interest. Literature review was conducted to compare PAGEANT with academic and industry tools. For 2504 genomes made publicly available through the 1000 Genomes Project, we derived their genomic characteristics for a suite of qualitative and quantitative traits. One exemplary trait is susceptibility to COVID-19, based on the most up-to-date scientific findings reported.

Control of posttranscriptional mRNA decay is a crucial determinant of cell homeostasis and differentiation. mRNA lifetime is governed by cis-regulatory elements in their 3' untranslated regions (UTR). Despite ongoing progress in the identification of cis elements we have little knowledge about the functional and structural integration of multiple elements in 3'UTR regulatory hubs and their recognition by mRNA-binding proteins (RBPs). Structural analyses are complicated by inconsistent mapping and prediction of RNA fold, by dynamics, and size. We here, for the first time, provide the secondary structure of a complete mRNA 3'UTR. We use NMR spectroscopy in a divide-and-conquer strategy complemented with SAXS, In-line probing and SHAPE-seq applied to the 3'UTR of Ox40 mRNA, which encodes a T-cell co-receptor repressed by the protein Roquin. We provide contributions of RNA elements to Roquin-binding. The protein uses its extended bi-modal ROQ domain to sequentially engage in a 2:1 stoichiometry with a 3'UTR core motif. We observe differential binding of Roquin to decay elements depending on their structural embedment. Our data underpins the importance of studying RNA regulation in a full sequence and structural context. This study serves as a paradigm for an approach in analysing structured RNA-regulatory hubs and their binding by RBPs.

Erratum in Nucleic Acids Res. 2022 Aug 26;50(15):8999.

Nucleic acid cleaving DNAzymes are versatile and robust catalysts that outcompete ribozymes and protein enzymes in terms of chemical stability, affordability and ease to synthesize. In spite of their attractiveness, the choice of which DNAzyme should be used to cleave a given substrate is far from obvious, and requires expert knowledge as well as in-depth literature scrutiny. DNAzymeBuilder enables fast and automatic assembly of DNAzymes for the first time, superseding the manual design of DNAzymes. DNAzymeBuilder relies on an internal database with information on RNA and DNA cleaving DNAzymes, including the reaction conditions under which they best operate, their kinetic parameters, the type of cleavage reaction that is catalyzed, the specific sequence that is recognized by the DNAzyme, the cleavage site within this sequence, and special design features that might be necessary for optimal activity of the DNAzyme. Based on this information and the input sequence provided by the user, DNAzymeBuilder provides a list of DNAzymes to carry out the cleavage reaction and detailed information for each of them, including the expected yield, reaction products and optimal reaction conditions. DNAzymeBuilder is a resource to help researchers introduce DNAzymes in their day-to-day research, and is publicly available at https://iimcb.genesilico.pl/DNAzymeBuilder.

We present ANANASTRA, https://ananastra.autosome.org, a web server for the identification and annotation of regulatory single-nucleotide polymorphisms (SNPs) with allele-specific binding events. ANANASTRA accepts a list of dbSNP IDs or a VCF file and reports allele-specific binding (ASB) sites of particular transcription factors or in specific cell types, highlighting those with ASBs significantly enriched at SNPs in the query list. ANANASTRA is built on top of a systematic analysis of allelic imbalance in ChIP-Seq experiments and performs the ASB enrichment test against background sets of SNPs found in the same source experiments as ASB sites but not displaying significant allelic imbalance. We illustrate ANANASTRA usage with selected case studies and expect that ANANASTRA will help to conduct the follow-up of GWAS in terms of establishing functional hypotheses and designing experimental verification.

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

pH regulates protein function and interactions by altering the charge of individual residues causing loss or gain of intramolecular noncovalent bonds, which may lead to structural rearrangements. While tools to analyze residue-specific charge distribution of proteins at a given pH exist, currently no tool is available to investigate noncovalent bond changes at two different pH values. To make protein pH sensitivity analysis more accessible, we developed patcHwork, a web server that combines the identification of amino acids undergoing a charge shift with the determination of affected noncovalent bonds at two user-defined pH values. At the sequence-only level, patcHwork applies the Henderson-Hasselbalch equation to determine pH-sensitive residues. When the 3D protein structure is available, patcHwork can be employed to gain mechanistic understanding of the effect of pH. This is achieved using the PDB2PQR and PROPKA tools and noncovalent bond determination algorithms. A user-friendly interface allows visualizing pH-sensitive residues, affected salt bridges, hydrogen bonds and aromatic (pi-pi and cation-pi) interactions. patcHwork can be used to identify patches, a new concept we propose of pH-sensitive residues in close proximity on the protein, which may have a major impact on function. We demonstrate the attractiveness of patcHwork studying experimentally investigated pH-sensitive proteins (https://patchwork.biologie.uni-freiburg.de/).

Deep learning techniques have significantly advanced the field of protein structure prediction. LOMETS3 (https://zhanglab.ccmb.med.umich.edu/LOMETS/) is a new generation meta-server approach to template-based protein structure prediction and function annotation, which integrates newly developed deep learning threading methods. For the first time, we have extended LOMETS3 to handle multi-domain proteins and to construct full-length models with gradient-based optimizations. Starting from a FASTA-formatted sequence, LOMETS3 performs four steps of domain boundary prediction, domain-level template identification, full-length template/model assembly and structure-based function prediction. The output of LOMETS3 contains (i) top-ranked templates from LOMETS3 and its component threading programs, (ii) up to 5 full-length structure models constructed by L-BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno algorithm) optimization, (iii) the 10 closest Protein Data Bank (PDB) structures to the target, (iv) structure-based functional predictions, (v) domain partition and assembly results, and (vi) the domain-level threading results, including items (i)-(iii) for each identified domain. LOMETS3 was tested in large-scale benchmarks and the blind CASP14 (14th Critical Assessment of Structure Prediction) experiment, where the overall template recognition and function prediction accuracy is significantly beyond its predecessors and other state-of-the-art threading approaches, especially for hard targets without homologous templates in the PDB. Based on the improved developments, LOMETS3 should help significantly advance the capability of broader biomedical community for template-based protein structure and function modelling.

WashU Epigenome Browser (https://epigenomegateway.wustl.edu/browser/) is a web-based genomic data exploration tool that provides visualization, integration, and analysis of epigenomic datasets. The newly renovated user interface and functions have enabled researchers to engage with the browser and genomic data more efficiently and effectively since 2018. Here, we introduce a new integrated panel design in the browser that allows users to interact with 1D (genomic features), 2D (such as Hi-C), 3D (genome structure), and 4D (time series) data in a single web page. The browser can display three-dimensional chromatin structures with the 3D viewer module. The 4D tracks, called 'Dynamic' tracks, animatedly display time-series data, allowing for a more striking visual impact to identify the gene or genomic region candidates as a function of time. Genomic data, such as annotation features, numerical values, and chromatin interaction data can all be viewed in the dynamic track mode. Imaging data from microscopy experiments can also be displayed in the browser. In addition to software development, we continue to service and expand the data hubs we host for large consortia including 4DN, Roadmap Epigenomics, TaRGET and ENCODE, among others. Our growing user/developer community developed additional track types as plugins, such as qBed and dynseq tracks, which extend the utility of the browser. The browser serves as a foundation for additional genomics platforms including the WashU Virus Genome Browser (for COVID-19 research) and the Comparative Genome Browser. The WashU Epigenome Browser can also be accessed freely through Amazon Web Services at https://epigenomegateway.org/.

3DLigandSite is a web tool for the prediction of ligand-binding sites in proteins. Here, we report a significant update since the first release of 3DLigandSite in 2010. The overall methodology remains the same, with candidate binding sites in proteins inferred using known binding sites in related protein structures as templates. However, the initial structural modelling step now uses the newly available structures from the AlphaFold database or alternatively Phyre2 when AlphaFold structures are not available. Further, a sequence-based search using HHSearch has been introduced to identify template structures with bound ligands that are used to infer the ligand-binding residues in the query protein. Finally, we introduced a machine learning element as the final prediction step, which improves the accuracy of predictions and provides a confidence score for each residue predicted to be part of a binding site. Validation of 3DLigandSite on a set of 6416 binding sites obtained 92% recall at 75% precision for non-metal binding sites and 52% recall at 75% precision for metal binding sites. 3DLigandSite is available at https://www.wass-michaelislab.org/3dligandsite. Users submit either a protein sequence or structure. Results are displayed in multiple formats including an interactive Mol* molecular visualization of the protein and the predicted binding sites.

The EMBL-EBI search and sequence analysis tools frameworks provide integrated access to EMBL-EBI's data resources and core bioinformatics analytical tools. EBI Search (https://www.ebi.ac.uk/ebisearch) provides a full-text search engine across nearly 5 billion entries, while the Job Dispatcher tools framework (https://www.ebi.ac.uk/services) enables the scientific community to perform a diverse range of sequence analysis using popular bioinformatics applications. Both allow users to interact through user-friendly web applications, as well as via RESTful and SOAP-based APIs. Here, we describe recent improvements to these services and updates made to accommodate the increasing data requirements during the COVID-19 pandemic.