Ribosomes are essential nanomachines responsible for all protein production in cells. Ribosome biogenesis and function are energy costly processes, they are tightly regulated to match cellular needs. In cancer, major pathways that control ribosome biogenesis and function are often deregulated to ensure cell survival and to accommodate the continuous proliferation of tumour cells. Ribosomal RNAs (rRNAs) are abundantly modified with 2'-O-methylation (Nm, ribomethylation) being one of the most common modifications. In eukaryotic ribosomes, ribomethylation is performed by the methyltransferase Fibrillarin guided by box C/D small nucleolar RNAs (snoRNAs). Accumulating evidences indicate that snoRNA expression and ribosome methylation profiles are altered in cancer. Here we review our current knowledge on differential snoRNA expression and rRNA 2'-O methylation in the context of human malignancies, and discuss the consequences and opportunities for cancer diagnostics, prognostics, and therapeutics.

RNA editing by cytosine and adenosine deaminases changes the identity of the edited bases. While cytosines are converted to uracils, adenines are converted to inosines. If coding regions of mRNAs are affected, the coding potential of the RNA can be changed, depending on the codon affected. The recoding potential of nucleotide deaminases has recently gained attention for their ability to correct genetic mutations by either reverting the mutation itself or by manipulating processing steps such as RNA splicing. In contrast to CRISPR-based DNA-editing approaches, RNA editing events are transient in nature, therefore reducing the risk of long-lasting inadvertent side-effects. Moreover, some RNA-based therapeutics are already FDA approved and their use in targeting multiple cells or organs to restore genetic function has already been shown. In this review, we provide an overview on the current status and technical differences of site-directed RNA-editing approaches. We also discuss advantages and challenges of individual approaches.

The covalent modification of RNA molecules is a pervasive feature of all classes of RNAs and has fundamental roles in the regulation of several cellular processes. Mapping the location of RNA modifications transcriptome-wide is key to unveiling their role and dynamic behaviour, but technical limitations have often hampered these efforts. Nanopore direct RNA sequencing is a third-generation sequencing technology that allows the sequencing of native RNA molecules, thus providing a direct way to detect modifications at single-molecule resolution. Despite recent advances, the analysis of nanopore sequencing data for RNA modification detection is still a complex task that presents many challenges. Many works have addressed this task using different approaches, resulting in a large number of tools with different features and performances. Here we review the diverse approaches proposed so far and outline the principles underlying currently available algorithms.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

5-methylcytosine (m5C) is identified as an abundant and conserved modification in various RNAs, including tRNAs, mRNAs, rRNAs, and other non-coding RNAs. The application of high-throughput sequencing and mass spectrometry allowed for the detection of m5C at a single-nucleotide resolution and at a global abundance separately; this contributes to a better understanding of m5C modification and its biological functions. m5C modification plays critical roles in diverse aspects of RNA processing, including tRNA stability, rRNA assembly, and mRNA translation. Notably, altered m5C modifications and mutated RNA m5C methyltransferases are associated with diverse pathological processes, such as nervous system disorders and cancers. This review may provide new sights of molecular mechanism and functional importance of m5C modification.

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.

Ribonucleoprotein (RNP) assemblies are ubiquitous in eukaryotic cells and have functions throughout RNA transcription, splicing, and stability. Of the RNA-binding proteins that form RNPs, TAR DNA-binding protein of 43 kD (TDP43) is of particular interest due to its essential nature and its association with disease. TDP43 plays critical roles in RNA metabolism, many of which require its recruitment to RNP granules such as stress granules, myo-granules, and neuronal transport granules. Moreover, the presence of cytoplasmic TDP43-positive inclusions is a pathological hallmark of several neurodegenerative diseases. Despite the pervasiveness of TDP43 aggregates, TDP43 mutations are exceedingly rare, suggesting that aggregation may be linked to dysregulation of TDP43 function. Oligomerization is a part of normal TDP43 function; thus, it is of interest to understand what triggers the irreversible aggregation that is seen in disease. Herein, we examine TDP43 functions, particularly in RNP granules, and the mechanisms which may explain pathological TDP43 aggregation.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex.Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

Considered to be a field that is continuously growing, epitranscriptomics analyzes the modifications that occur in RNA transcripts and their downstream effects. As epigenetic modifications found in DNA and histones exhibit specific roles on various biological processes, also epitranscriptomic marks control gene expression patterns that are crucial for proper cell proliferation, differentiation and tissue development. Thus, various epitranscriptomic signatures have been identified to play specific roles during stem cell differentiation towards the neuronal and glial lineages, axonal guidance, synaptic plasticity, thus leading to the development of the mature brain tissue. Here we describe in-depth molecular mechanism underlying the most important RNA modifications with emerging roles in the nervous system.

In the cell, RNA abundance is dynamically controlled by transcription and decay rates. Posttranscriptional nucleotide addition at the RNA 3' end is a means of regulating mRNA and RNA stability and activity, as well as marking RNAs for degradation. The human nucleotidyltransferase Gld2 polyadenylates mRNAs and monoadenylates microRNAs, leading to an increase in RNA stability. The broad substrate range of Gld2 and its role in controlling RNA stability make the regulation of Gld2 activity itself imperative. Gld2 activity can be regulated by post-translational phosphorylation via the oncogenic kinase Akt1 and other kinases, leading to either increased or almost abolished enzymatic activity, and here we confirm that Akt1 phosphorylates Gld2 in a cellular context. Another means to control Gld2 RNA specificity and activity is the interaction with RNA binding proteins. Known interactors are QKI-7 and CPEB, which recruit Gld2 to specific miRNAs and mRNAs. We investigate the interplay between five phosphorylation sites in the N-terminal domain of Gld2 and three RNA binding proteins. We found that the activity and RNA specificity of Gld2 is dynamically regulated by this network. Binding of QKI-7 or phosphorylation at S62 relieves the autoinhibitory function of the Gld2 N-terminal domain. Binding of QKI-7 to a short peptide sequence within the N-terminal domain can also override the deactivation caused by Akt1 phosphorylation at S116. Our data revealed that Gld2 substrate specificity and activity can be dynamically regulated to match the cellular need of RNA stabilization and turnover.

The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAsAsp,Asn,His,Tyr. While both subunits of the functional heterodimer have been crystallized individually, much of our understanding of its dimer interface or recognition of a target RNA has been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ1, deviates not only in function, but as a homodimer, also in its subunit architecture, any inferences regarding the subunit association of the eukaryotic heterodimer or the significance of its unique catalytically inactive subunit are based on unstable footing. Here, we report the crystal structure of human TGT in its heterodimeric form and in complex with a 25-mer stem loop RNA, enabling detailed analysis of its dimer interface and interaction with a minimal substrate RNA. Based on a model of bound tRNA, we addressed a potential functional role of the catalytically inactive subunit QTRT2 by UV-crosslinking and mutagenesis experiments, identifying the two-stranded βEβF-sheet of the QTRT2 subunit as an additional RNA-binding motif.

The blood-brain barrier (BBB), which controls permeability into and out of the nervous system, is a tightly connected, structural, and functional separation between the central nervous system (CNS) and circulating blood. CNS diseases, such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, stroke, meningitis, and brain cancers, often develop with the increased BBB permeability and further leads to irreversible CNS injury. Non-coding RNAs (ncRNAs) are functional RNA molecules that generally lack the coding abilities but can actively regulate the mRNA expression and function through different mechanisms. Various types of ncRNAs, including microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs), are highly expressed in brain microvascular endothelial cells and are potential mediators of BBB permeability. Here, we summarized the recent research progress on miRNA, lncRNA, and circRNA roles regulating the BBB permeability in different CNS diseases. Understanding how these ncRNAs affect the BBB permeability shall provide important therapeutic insights into the prevention and control of the BBB dysfunction.

RNA editing generates genetic diversity in mammals by altering amino acid sequences, miRNA targeting site sequences, influencing the stability of targeted RNAs, and causing changes in gene expression. However, the extent to which RNA editing affect gene expression via modifying miRNA binding site remains unexplored. Here, we first profiled the dynamic A-to-I RNA editome across tissues of Duroc and Luchuan pigs. The RNA editing events at the miRNA binding sites were generated. The biological function of the differentially edited gene in skeletal muscle was further characterized in pig muscle-derived satellite cells. RNA editome analysis revealed a total of 171,909 A-to-I RNA editing sites (RESs), and examination of its features showed that these A-to-I editing sites were mainly located in SINE retrotransposons PRE-1/Pre0_SS element. Analysis of differentially edited sites (DESs) revealed a total of 4,552 DESs across tissues between Duroc and Luchuan pigs, and functional category enrichment analysis of differentially edited gene (DEG) sets highlighted a significant association and enrichment of tissue-developmental pathways including TGF-beta, PI3K-Akt, AMPK, and Wnt signaling pathways. Moreover, we found that RNA editing events at the miRNA binding sites in the 3'-UTR of HSPA12B mRNA could prevent the miRNA-mediated mRNA downregulation of HSPA12B in the muscle-derived satellite (MDS) cell, consistent with the results obtained from the Luchuan skeletal muscle. This study represents the most systematic attempt to characterize the significance of RNA editing in regulating gene expression, particularly in skeletal muscle, constituting a new layer of regulation to understand the genetic mechanisms behind phenotype variance in animals.Abbreviations: A-to-I: Adenosine-to-inosine; ADAR: Adenosine deaminase acting on RNA; RES: RNA editing site; DEG: Differentially edited gene; DES: Differentially edited site; FDR: False discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopaedia of Genes and Genomes; MDS cell: musclederived satellite cell; RPKM: Reads per kilobase of exon model in a gene per million mapped reads; UTR: Untranslated coding regions.

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

Many long noncoding RNAs (lncRNAs) are localized in the nucleus and play important roles in various biological processes, including cell proliferation, differentiation and antiviral response. Yet, it remains unclear how some nuclear lncRNAs are turned over. Here we show that the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) controls expression levels of NEAT1v2, a lncRNA involved in the formation of nuclear paraspeckles. hnRNPH1 associates, in an RNA-independent manner, with the RNA helicase MTR4/MTREX, an essential co-factor of the nuclear ribonucleolytic RNA exosome. hnRNPH1 localizes in nuclear speckles and depletion of hnRNPH1 enhances NEAT1v2-mediated expression of the IL8 mRNA, encoding a cytokine involved in the innate immune response. Taken together, our results indicate that the hnRNPH1-MTR4 linkage regulates IL8 expression through the degradation of NEAT1v2 RNA.

Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

Gemin5 is a multifaceted RNA-binding protein that comprises distinct structural domains, including a WD40 and TPR-like for which the X-ray structure is known. In addition, the protein contains a non-canonical RNA-binding domain (RBS1) towards the C-terminus. To understand the RNA binding features of the RBS1 domain, we have characterized its structural characteristics by solution NMR linked to RNA-binding activity. Here we show that a short version of the RBS1 domain that retains the ability to interact with RNA is predominantly unfolded even in the presence of RNA. Furthermore, an exhaustive mutational analysis indicates the presence of an evolutionarily conserved motif enriched in R, S, W, and H residues, necessary to promote RNA-binding via π-π interactions. The combined results of NMR and RNA-binding on wild-type and mutant proteins highlight the importance of aromatic and arginine residues for RNA recognition by RBS1, revealing that the net charge and the π-amino acid density of this region of Gemin5 are key factors for RNA recognition.

RNA molecules are known to fold into specific structures which often play a central role in their functions and regulation. In silico folding of RNA transcripts, especially when assisted with structure profiling (SP) data, is capable of accurately elucidating relevant structural conformations. However, such methods scale poorly to the swaths of SP data generated by transcriptome-wide experiments, which are becoming more commonplace and advancing our understanding of RNA structure and its regulation at global and local levels. This has created a need for tools capable of rapidly deriving structural assessments from SP data in a scalable manner. One such tool we previously introduced that aims to process such data is patteRNA, a statistical learning algorithm capable of rapidly mining big SP datasets for structural elements. Here, we present a reformulation of patteRNA's pattern recognition scheme that sees significantly improved precision without major compromises to computational overhead. Specifically, we developed a data-driven logistic classifier which interprets patteRNA's statistical characterizations of SP data in addition to local sequence properties as measured with a nearest neighbour thermodynamic model. Application of the classifier to human structurome data reveals a marked association between detected stem-loops and RNA binding protein (RBP) footprints. The results of our application demonstrate that upwards of 30% of RBP footprints occur within loops of stable stem-loop elements. Overall, our work arrives at a rapid and accurate method for automatically detecting families of RNA structure motifs and demonstrates the functional relevance of identifying them transcriptome-wide.

Epitranscriptomic modifications of stable RNAs are dynamically regulated and specific profiles of 2'-O-methylation in rRNA have been associated with distinct cancer types. However, these observations pointed out the existence of at least two distinct groups: a rather large group with constitutive rRNA Nm residues exhibiting a stable level of methylation and a more restricted set of variable modifications, giving rise to the concept of 'specialized ribosomes'. These heterogeneous ribosomes can modulate their translational properties and be key regulatory players, depending on the physiological state of the cell. However, these conclusions were drawn from a limited set of explored human cell lines or tissues, mostly related to cancer cells of the same type. Here, we report a comprehensive analysis of human rRNA Nm modification variability observed for >15 human cell lines grown in different media and conditions. Our data demonstrate that human Nm sites can be classified into four groups, depending on their observed variability. About ⅓ of rRNA 2'-O-methylations are almost invariably modified at the same level in all tested samples (stable modifications), the second group of relatively invariant modifications (another ½ of the total) showing a slightly higher variance (low variable group) and two variable groups, showing an important heterogeneity. Mapping of these four classes on the human ribosome 3D structure shows that stably modified positions are preferentially located in the important ribosome functional sites, while variable and highly variable residues are mostly distributed to the ribosome periphery. Possible relationships of such stable and variable modifications to the ribosome functions are discussed.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

Chimeric RNAs are gaining more and more attention as they have broad implications in both cancer and normal physiology. To date, over 40 chimeric RNA prediction methods have been developed to facilitate their identification from RNA sequencing data. However, a limited number of studies have been conducted to compare the performance of these tools; additionally, previous studies have become outdated as more software tools have been developed within the last three years. In this study, we benchmarked 16 chimeric RNA prediction software, including seven top performers in previous benchmarking studies, and nine that were recently developed. We used two simulated and two real RNA-Seq datasets, compared the 16 tools for their sensitivity, positive prediction value (PPV), F-measure, and also documented the computational requirements (time and memory). We noticed that none of the tools are inclusive, and their performance varies depending on the dataset and objects. To increase the detection of true positive events, we also evaluated the pair-wise combination of these methods to suggest the best combination for sensitivity and F-measure. In addition, we compared the performance of the tools for the identification of three classes (read-through, inter-chromosomal and intra-others) of chimeric RNAs. Finally, we performed TOPSIS analyses and ranked the weighted performance of the 16 tools.

In Drosophila melanogaster, PD isoform of the double-stranded RNA binding protein (dsRBP) Loquacious (Loqs-PD) facilitates dsRNA cleavage to siRNA by Dicer-2. StaufenC (StauC) was discovered as a coleopteran-specific dsRBP required for dsRNA processing in coleopteran insects. Here, we show that StauC is essential for the high RNAi efficiency observed in coleopterans. Knockdown of StauC but not the homologs of Loqs-PD and R2D2 evoked a long-lasting insensitivity to RNAi in the coleopteran cell line, Ledp-SL1. The dsRNA insensitivity induced by StauC knockdown could not be overcome merely by an increase in dose or time of exposure to dsRNA or expression of Loquacious or R2D2. Furthermore, StauC but not Loqs and R2D2 are required for processing of dsRNA into siRNA. StauC overexpression also partly restored the impaired RNAi caused by the knockdown of Loqs-PD in D. melanogaster Kc cells. However, StauC was unable to compensate for the loss-of-the function of Dcr-2 or R2D2. Overall, these data suggest that StauC functions like Lops-PD in processing dsRNA to siRNA.

ARF: alternative reading frame, that is, p14ARF, or CDKN2A (cyclin-dependent kinase inhibitor 2A); β-gal: β-galactosidase; CLIP-seq: crosslinking and immunoprecipitation-sequencing; DMTF1: the cyclin D binding myb-like transcription factor 1; ESS/ESE: exonic splicing silencer/enhancer; Ex: exon; FBS: fetal bovine serum; Gluc: Gaussia luciferase; hnRNPs: heterogeneous nuclear ribonucleoproteins; In: intron; ISS/ISE: intronic splicing silencer/enhancer; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PSI: percent-splice-in; qPCR: quantitative real-time PCR; RIP: RNA immunoprecipitation; RNAseq: RNA sequencing; RT: reverse transcription; SF1: splicing factor 1; SR: serine/arginine-rich proteins; SRSF5: serine and arginine-rich splicing factor 5; TCGA: the cancer genome atlas; UCSC: University of California, Santa Cruz. WT: Wild type.

RNA-seq has been widely used to reveal the molecular mechanism of variants of life process. We have developed an alternative method, MustSeq, which generates multiple second strands along a single 1st strand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3' ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5'-end capture, enables early pooling of multiple samples, and requires only one twentieth of sequencing reads of full-length sequencing. We demonstrate the power and features of MustSeq comparing with TruSeq and NEBNext RNA-seq, two conventional full-length methods and QuantSeq, an industrial 3' end method. In cancer cell lines, the reads distribution of CDS-exon as well as genes, lncRNAs and GO terms detected by MustSeq are closer than QuantSeq to TruSeq. In mouse hepatocarcinoma and healthy livers, MustSeq enriches the same pathways as by NEBNext, and reveals the molecular profile of carcinogenesis. Overall MustSeq is a robust and accurate RNA-seq method allowing efficient library construction, sequencing and analysis, particularly valuable for analysis of differentially expressed genes with a large number of samples. MustSeq will greatly accelerate the application of bulk RNA-seq on different fields, and potentially applicable for single cell RNA-seq.

The contributions of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) to breast cancer are critical areas of investigation. In this study, we identified a novel lncRNA RP11-283G6.5 which was lowly expressed in breast cancer and whose low expression was correlated with poor overall survival and disease-free survival of breast cancer patients. Functional experiments revealed that ectopic expression of RP11-283G6.5 confined breast cancer cellular growth, migration, and invasion, and promoted cellular apoptosis. Conversely, RP11-283G6.5 silencing facilitated breast cancer cellular growth, migration, and invasion, and repressed cellular apoptosis. Moreover, RP11-283G6.5 was found to confine breast cancer tumour growth and metastasis in vivo. Mechanistically, RP11-283G6.5 competitively bound to ILF3, reduced the binding of ILF3to primary miR-188 (pri-miR-188), abolished the suppressive effect of ILF3 on pri-miR-188 processing, and therefore promoted pri-miR-188 processing, leading to the reduction of pri-miR-188 and the upregulation of mature miR-188-3p. The expression of RP11-283G6.5 was significantly positively correlated with that of miR-188-3p in breast cancer tissues. Through increasing miR-188-3p, RP11-283G6.5 decreased TMED3, a target of miR-188-3p. RP11-283G6.5 further suppressed Wnt/β-catenin signalling via decreasing TMED3. Rescue assays revealed that inhibition of miR-188-3p, overexpression of TMED3 or blocking Wnt/β-catenin signalling all attenuated the roles of RP11-283G6.5 in breast cancer. Collectively, these findings demonstrated that RP11-283G6.5 is a tumour suppressive lncRNA in breast cancer via modulating miR-188-3p/TMED3/Wnt/β-catenin signalling. This study indicated that RP11-283G6.5 might be a promising prognostic biomarker and therapeutic target for breast cancer.

Biochemical studies of the human ribosome synthesis pathway have been hindered by technical difficulties in obtaining intact preribosomal complexes from internal regions of the nucleolus. Here we provide a detailed description of an extraction method that enables efficient detection, isolation, and characterization of nucleolar preribosomes containing large pre-rRNA species. The three-step Preribosome Sequential Extraction (PSE) protocol preserves the integrity of early preribosomal complexes and yields preparations amenable to biochemical analyses from low amounts of starting material. We validate this procedure through the detection of specific trans-acting factors and pre-rRNAs in the extracted preribosomes using affinity matrix pull-downs and sedimentation assays. In addition, we describe the application of the PSE method for monitoring cellular levels of ribosome-free 5S RNP complexes as an indicator of ribosome biogenesis stress. Our optimized experimental procedures will facilitate studies of human ribosome biogenesis in normal, mutant and stressed-cell scenarios, including the characterization of candidate ribosome biogenesis factors, preribosome interactors under specific physiological conditions or effects of drugs on ribosome maturation.

Circular RNAs (circRNAs) are regulatory molecules involved in the modulation of gene expression. Although originally assumed as non-coding RNAs, recent studies have evidenced that animal circRNAs can act as translatable transcripts. The study of plant-circRNAs is incipient, and no autonomous coding plant-circRNA has been described yet. Viroids are the smallest plant-pathogenic circRNAs known to date. Since their discovery 50 years ago, viroids have been considered valuable systems for the study of the structure-function relationships in RNA, essentially because they have not been shown to have coding capacity. We used two pathogenic circRNAs (Hop stunt viroid and Eggplant latent viroid) as experimental tools to explore the coding potential of plant-circRNAs. Our work supports that the analysed viroids contain putative ORFs able to encode peptides carrying subcellular localization signals coincident with the corresponding replication-specific organelle. Bioassays in well-established hosts revealed that mutations in these ORFs diminish their biological efficiency. Interestingly, circular forms of HSVd and ELVd were found to co-sediment with polysomes, revealing their physical interaction with the translational machinery of the plant cell. Based on this evidence we hypothesize about the possibility that plant circRNAs in general, and viroids in particular, can act, under certain cellular conditions, as non-canonical translatable transcripts.

The high-resolution feature of single-cell transcriptome sequencing technology allows researchers to observe cellular gene expression profiles at the single-cell level, offering numerous possibilities for subsequent biomedical investigation. However, the unavoidable technical impact of high missing values in the gene-cell expression matrices generated by insufficient RNA input severely hampers the accuracy of downstream analysis. To address this problem, it is essential to develop a more rapid and stable imputation method with greater accuracy, which should not only be able to recover the missing data, but also effectively facilitate the following biological mechanism analysis. The existing imputation methods all have their drawbacks and limitations, some require pre-assumed data distribution, some cannot distinguish between technical and biological zeros, and some have poor computational performance. In this paper, we presented a novel imputation software FRMC for single-cell RNA-Seq data, which innovates a fast and accurate singular value thresholding approximation method. The experiments demonstrated that FRMC can not only precisely distinguish 'true zeros' from dropout events and correctly impute missing values attributed to technical noises, but also effectively enhance intracellular and intergenic connections and achieve accurate clustering of cells in biological applications. In summary, FRMC can be a powerful tool for analysing single-cell data because it ensures biological significance, accuracy, and rapidity simultaneously. FRMC is implemented in Python and is freely accessible to non-commercial users on GitHub: https://github.com/HUST-DataMan/FRMC.

MiRNA isoforms (isomiRs) are single stranded small RNAs originating from the same pri-miRNA hairpin as a result of cleavage by Drosha and Dicer enzymes. Variations at the 5'-end of a miRNA alter the seed region of the molecule, thus affecting the targetome of the miRNA. In this manuscript, we analysed the distribution of miRNA cleavage positions across 31 different cancers using miRNA sequencing data of TCGA project. As a result, we found that the processing positions are not tissue specific and that all miRNAs could be correctly classified as ones exhibiting homogeneous or heterogeneous cleavage at one of the four cleavage sites. In 42% of cases (42 out of 100 miRNAs), we observed imprecise 5'-end Dicer cleavage, while this fraction was only 14% for Drosha (14 out of 99). To the contrary, almost all cleavage sites of 3'-ends (either Drosha or Dicer) were heterogeneous. With the use of only four nucleotides surrounding a 5'-end Dicer cleavage position we built a model which allowed us to distinguish between homogeneous and heterogeneous cleavage with the reliable quality (ROC AUC = 0.68). Finally, we showed the possible applications of the study by the analysis of two 5'-end isoforms originating from the same exogeneous shRNA hairpin. It turned out that the less expressed shRNA variant was functionally active, which led to the increased off-targeting. Thus, the obtained results could be applied to the design of shRNAs whose processing will result in a single 5'-variant.

Melanoma is considered as the most frequent primary malignancy occurring in skin. Accumulating studies have suggested that long non-coding RNAs (lncRNAs) play critical parts in multiple cancers. In this study, we explored the molecular mechanism of ZBED3 antisense RNA 1 (ZBED3-AS1) in melanoma. We observed that ZBED3-AS1 expression was remarkably up-regulated in melanoma tissues, and high ZBED3-AS1 level was linked to unsatisfactory survival of melanoma patients. Then, we discovered that ZBED3-AS1 was overexpressed in melanoma cells compared with human epidermal melanocytes. In addition, loss-of-function assays verified that ZBED3-AS1 knockdown restrained cell proliferation, migration, epithelial-mesenchymal transition (EMT), and stemness in melanoma. In addition, signal transducer and activator of transcription 3 (STAT3), which also showed tumour-facilitating functions in melanoma, was confirmed as a transcriptional activator of ZBED3-AS1. Moreover, ZBED3-AS1 enhanced the expression of AT-rich interaction domain 4B (ARID4B) through sequestering miR-381-3p. Importantly, we further confirmed that ZBED3-AS1 promoted the malignant progression of melanoma by regulating miR-381-3p/ARID4B axis to activate the phosphatidylinositol 3-kinase/AKT serine/threonine kinase (PI3K/AKT) signalling pathway. In a word, our research might provide a novel therapeutic target for melanoma.

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.

The expression and localization of the oncoprotein c-Myc is highly regulated at the level of transcription, mRNA transport, translation, as well as stability of the protein. We previously showed that Annexin A2 (AnxA2) binds to a specific localization element in the 3'untranslated region (UTR) of c-myc mRNA and is involved in its localization to the perinuclear region. In the present study, we demonstrate that AnxA2 binds in a Ca2+-dependent manner to the internal ribosomal entry site (IRES) containing two pseudo-knots in the 5´UTR of the c-myc mRNA. Here, we employ an in vitro rabbit reticulocyte lysate system with chimeric c-myc reporter mRNAs to demonstrate that binding of AnxA2 to the c-myc IRES modulates the expression of c-Myc. Notably, we show that low levels of AnxA2 appear to increase, while high levels of AnxA2 inhibits translation of the chimeric mRNA. However, when both the AnxA2-binding site and the ribosomal docking site in the c-myc IRES are deleted, AnxA2 has no effect on the translation of the reporter mRNA. Forskolin-treatment of PC12 cells results in upregulation of Ser25 phosphorylated AnxA2 expression while c-Myc expression is down-regulated. The effect of forskolin on c-Myc expression and the level of Ser25 phosphorylated AnxA2 was abolished in the presence of EGTA. These findings indicate that AnxA2 regulates both the transport and subsequent translation of the c-myc mRNA, possibly by silencing the mRNA during its transport. They also suggest that AnxA2 act as a switch to turn off the c-myc IRES activity in the presence of calcium.Abbreviations: AnxA2, Annexin A2; β2--µglob, β2-microglobulin; cpm, counts per minute; hnRNP, heterogenous nuclear ribonucleoprotein; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; MM, multiple myeloma; PABP, poly(A)-binding protein; PCBP, poly(rC) binding protein; PSF, PTB-associated splicing factor; PTB, polypyrimidine tract binding protein; RRL, rabbit reticulocyte lysate; UTR, untranslated region; YB, Y-box binding protein.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNA structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their downstream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs, we demonstrate first the significant association of an SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.

MicroRNAs (miRNAs) are increasingly seen as important regulators of placental development and opportunistic biomarker targets. Given the difficulty in obtaining samples from early gestation and subsequent paucity of the same, investigation of the role of miRNAs in early gestation human placenta has been limited. To address this, we generated miRNA profiles using 96 placentas from presumed normal pregnancies, across early gestation, in combination with matched profiles from maternal plasma. Placenta samples range from 6 to 23 weeks' gestation, a time period that includes placenta from the early, relatively low but physiological (6-10 weeks' gestation) oxygen environment, and later, physiologically normal oxygen environment (11-23 weeks' gestation).We identified 637 miRNAs with expression in 86 samples (after removing poor quality samples), showing a clear gestational age gradient from 6 to 23 weeks' gestation. We identified 374 differentially expressed (DE) miRNAs between placentas from 6-10 weeks' versus 11-23 weeks' gestation. We see a clear gestational age group bias in miRNA clusters C19MC, C14MC, miR-17 ~ 92 and paralogs, regions that also include many DE miRNAs. Proportional change in expression of placenta-specific miRNA clusters was reflected in maternal plasma.The presumed introduction of oxygenated maternal blood into the placenta (between ~10 and 12 weeks' gestation) changes the miRNA profile of the chorionic villus, particularly in placenta-specific miRNA clusters. Data presented here comprise a clinically important reference set for studying early placenta development and may underpin the generation of minimally invasive methods for monitoring placental health.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have been increasingly reported to serve vital parts in malignancies including CRC. Although cancer susceptibility 21 (CASC21) has been uncovered to play a part in CRC, its mechanism still needs further explanation. Thus, our study aimed to further explore the influence and mechanism of CASC21 in CRC progression. Quantitative real-time RT-PCR and western blot were performed to detect gene expression; a series of functional assays were performed to investigate the effect of CASC21 on CRC cells; in vivo tumour growth was evaluated via the nude mice xenograft model. The results revealed that CASC21 facilitated CRC cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness. In addition, CASC21 was co-expressed with and bound to transcription factor POU5F1B (POU class 5 homeobox 1B). CASC21 recruited POU5F1B to HGH1 promoter to activate the transcription of HGH1 homolog. Also, CASC21 served as a competitive endogenous RNA (ceRNA) to up-regulate HGH1 via endogenously sponging miR-485-5p. Moreover, HGH1 overexpression counteracted the suppression of CASC21 deficiency on CRC tumour growth. In summary, our study indicated that CASC21 enhanced the expression of HGH1 to promote the malignancy of CRC by recruiting POU5F1B and sponging miR-485-5p, suggesting a key role of CASC21 in CRC progression.

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

The human genome contains 481 ultraconserved regions (UCRs), which are genomic stretches of over 200 base pairs conserved among human, rat, and mouse. The majority of these regions are transcriptionally active (T-UCRs), and several have been found to be differentially expressed in tumours. Some T-UCRs have been functionally characterized, but of those few have been associated to breast cancer (BC). Using TCGA data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with oestrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. The expression levels of 12 T-UCRs were further analysed in a cohort of 82 Brazilian BC patients using RT-qPCR. We found that uc.147 is high expressed in luminal A and B patients. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is located in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic trancript, and its sequence was confirmed by RACE. The silencing of uc.147 increases apoptosis, arrests cell cycle, and reduces cell viability and colony formation in BC cell lines. Additionally, we identifed 19 proteins that interact with lnc-uc.147 through mass spectrometry and demonstrated a high correlation of lnc-uc.147 with the neighbour gene expression and miR-18 and miR-190b. This is the first study to analyse the expression of all T-UCRs in BC and to functionally assess the lnc-uc.147.

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3'-5' exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.