Nanopore long-read sequencing enables real-time monitoring and controlling of individual nanopores. This allows to enrich or deplete specific sequences in DNA sequencing in a process called "adaptive sampling". So far, adaptive sampling was not applicable to direct sequencing of RNA. Here, we show that adaptive sampling is feasible and useful for direct RNA sequencing, which has its specific technical and biological challenges. Employing a well-controlled in vitro transcript-based model system, we identify essential characteristics and parameter settings for adaptive sampling in direct RNA sequencing, as the superior performance of depletion over enrichment. Here, the efficiency of depletion is close to the theoretical maximum. Additionally, we demonstrate that adaptive sampling efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our adaptive sampling approach to polyA-enriched RNA samples from human induced pluripotent stem cell-derived cardiomyocytes and mouse whole heart tissue and show efficient 2.5 to 2.8 fold depletion of highly abundant mitochondrial-encoded transcripts. Finally, we characterize depletion and enrichment performance for complex transcriptome subsets i.e. at the level of the entire Chromosome 11, proving the general applicability of direct RNA adaptive sampling. Our analyses provide evidence that adaptive sampling is especially useful to enable detection of lowly expressed transcripts and reduce the sequencing of highly abundant disturbing transcripts. Workflow and sequencing data are provided on Zenodo: https://doi.org/10.5281/zenodo.7701823.

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In S. cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1 and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation by studying the Escherichia coli thiB thiamine pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the anti-sequestering stem that could mediates the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the anti-sequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the anti-sequestering stem break or bias thiB function according to whether the anti-sequestering stem, or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (Homolog of Aquifex RNase P). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene rnpB) and protein (gene rnpA) subunits of RNP RNase P, but only the HARP of the thermophile Thermodesulfatator indicus (phylum Thermodesulfobacteria) was found to have robust tRNA 5'-end maturation activity in vitro and in vivo in an Escherichia coli RNase P depletion strain. These findings suggest that both types of RNase P are able to contribute to the essential tRNA 5'-end maturation activity in T. indicus, thus resembling the predicted evolutionary transition state in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes in this family of bacteria. Remarkably, T. indicus RNase P RNA is transcribed with a P12 expansion segment that is post-transcriptionally excised in vivo, such that the major fraction of the RNA is fragmented and thereby truncated by ~70 nt in the native T. indicus host as well as in the E. coli complementation strain. Replacing the native P12 element of T. indicus RNase P RNA with the short P12 helix of Thermotoga maritima RNase P RNA abolished fragmentation, but simultaneously impaired complementation efficiency in E. coli cells, suggesting that intracellular fragmentation and truncation of T. indicus RNase P RNA may be beneficial to RNA folding and/or enzymatic activity.

The modern ribosome catalyzes all coded protein synthesis in extant organisms. It is likely that its core structure is a direct descendant from the ribosome present in the last common ancestor (LCA). Hence, its earliest origins likely predate the LCA and therefore date further back in time. Of special interest is the pseudo-symmetrical region (SymR) that lies deep within the large subunit (LSU) where the peptidyl transfer reaction takes place. It was previously proposed that two RNA oligomers, representing the P- and A-regions of extant ribosomes dimerized to create a pore-like structure, which hosted the necessary properties, that facilitate peptide bond formation. However, recent experimental studies show that this may not be the case. Instead, several RNA constructs derived exclusively from the P-region were showed to form a homodimer capable of peptide bond synthesis. This seemingly modest change in our understanding is important because it provides a deeper understanding of ribosome's history. In practice this will change the focus of future research, which will likely increasingly examine the origins and properties of the P-region. Of special interest will be the origin issues as the homodimer would have allowed a pre-LCA ribosome that was significantly smaller than previously proposed. For the A-region the immediate issue will likely be its origin and whether it enhances ribosome performance. Here, we re-analyze the RNA/RNA interaction regions that most likely lead to SymR formation in the light of these recent findings. Further, it has been suggested that the ability of these RNA constructs to dimerize and enhance peptide bond formation is sequence dependent. We have analyzed the implications of sequence variations as parts of functional and non-functional constructs.

Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the immune response. Here, we review how Z-RNA features present in cellular RNAs -especially containing retroelements- could be recognized by a family of winged helix proteins, with an impact on host defense. We also discuss how mutations to specific Z-contacting amino acids disrupt their ability to stabilize Z-RNA, resulting in functional losses. We end by highlighting knowledge gaps in the field, which, if addressed, would significantly advance this active area of research.

Alternative splicing (AS) of mRNAs is an essential regulatory mechanism in eukaryotic gene expression. AS misregulation, caused by either dysregulation or mutation of splicing factors, has been shown to be involved in cancer development and progression, making splicing factors suitable targets for cancer therapy. In recent years, various types of pharmacological modulators, such as small molecules and oligonucleotides, targeting distinct components of the splicing machinery, have been under development to treat multiple disorders. Although these approaches have promise, targeting the core spliceosome components disrupts the early stages of spliceosome assembly and can lead to non-specific and toxic effects. New research directions have been focused on targeting specific splicing factors for a more precise effect. In this perspective, we will highlight several approaches for targeting splicing factors and their functions and suggest ways to improve their specificity.

In this short Perspective, we discuss the history of, and recent progress toward, the development of aptamers that can serve as rapid onset anticoagulants during cardiopulmonary bypass (CPB), extracorporeal membrane oxygenation (ECMO), and catheter-based diagnostic and interventional procedures, several million of which are performed each year worldwide. Aptamer anticoagulants provide potent and antidote-controllable anticoagulation and have low immunogenicity. New methods of aptamer isolation and engineering have not only improved the quality of aptamers, but also accelerated their development. Unfortunately, no aptamer identified to date can produce an anticoagulant effect as potent as that produced by unfractionated heparin (UFH), the standard anticoagulant for CPB. We have suggested several possible strategies to amplify the anticoagulant potency of existing aptamer anticoagulants.

Disruptions in RNA processing play critical roles in the pathogenesis of neurological diseases. In this perspective, we discuss recent progress in the development of RNA-targeting therapeutic modalities. We focus on progress, limitations, and opportunities in a new generation of therapies engineered from RNA binding proteins and other endogenous RNA regulatory macromolecules to treat human neurological disorders.

RNA structures regulate a wide range of processes in biology and disease, yet small molecule chemical probes or drugs that can modulate these functions are rare. Machine learning and other computational methods are well poised to fill gaps in knowledge and overcome the inherent challenges in RNA targeting, such as the dynamic nature of RNA and the difficulty of obtaining RNA high-resolution structures. Successful tools to date include principal component analysis, linear discriminate analysis, k-nearest neighbor, artificial neural networks, multiple linear regression, and many others. Employment of these tools has revealed critical factors for selective recognition in RNA:small molecule complexes, predictable differences in RNA and protein binding ligands, and quantitative structure activity relationships that allow the rational design of small molecules for a given RNA target. Herein we present our perspective on the value of using machine learning and other computation methods to advance RNA: small molecule targeting, including select examples and their validation as well as necessary and promising future directions that will be key to accelerate discoveries in this important field.

In the last decade, the field of oligonucleotide therapeutics has matured, with the regulatory approval of several single stranded and double stranded RNA drugs. In this perspective, I discuss enabling developments and likely future directions in the field from the perspective of oligonucleotide chemistry.

Adenosine deamination by the ADAR family of enzymes is a natural process that edits genetic information as it passes through messenger RNA. Adenosine is converted to inosine in mRNAs, and this base is interpreted as guanosine during translation. Realizing the potential of this activity for therapeutics, a number of researchers have developed systems that redirect ADAR activity to new targets, ones that aren't normally edited. These site-directed RNA editing (SDRE) systems can be broadly classified into two categories: ones that deliver an antisense RNA oligonucleotide to bind opposite a target adenosine, creating an editable structure that endogenously expressed ADARs recognize, and ones that tether the catalytic domain of recombinant ADAR to an antisense RNA oligonucleotide that serves as a targeting mechanism, much like with CRISPR-Cas or RNAi. To date, SDRE has been used mostly to try and correct genetic mutations. Here we argue that these applications are not ideal SDRE, mostly because RNA edits are transient and genetic mutations are not. Instead, we suggest that SDRE could be used to tune cell physiology to achieve temporary outcomes that are therapeutically advantageous, particularly in the nervous system. These include manipulating excitability in nociceptive neural circuits, abolishing specific phosphorylation events to reduce protein aggregation related to neurodegeneration or reduce the glial scarring that inhibits nerve regeneration, or enhancing G protein-coupled receptor signaling to increase nerve proliferation for the treatment of sensory disorders like blindness and deafness.

With over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a non-toxic manner.

Splice modulating antisense oligonucleotides (ASOs) offer treatment options for rare neurological diseases, including those with very rare mutations, where patient-specific, individualized ASOs have to be developed. Inspired by the development of milasen, the 1 Mutation 1 Medicine (1M1M) and Dutch Center for RNA Therapeutics (DCRT) aim to develop patient-specific ASOs and treat eligible patients within Europe and the Netherlands, respectively. Treatment will be provided under a named patient setting. Our initiatives benefited from regulatory advice from the European Medicines Agency (EMA) with regards to preclinical proof-of-concept studies, safety studies, compounding and measuring benefit and safety in treated patients. We here outline the most important considerations from these interactions and how we implemented this advice into our plan to develop and treat eligible patients within Europe.

RNAi interference (RNAi) is almost always associated with inhibition of translation in cell cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs.

RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson crick (WC) base pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs) have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl) glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance-based mechanism. In the last three decades various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and non-coding RNAs for a myriad of therapeutic applications.

p53 protein is a key regulator of cellular homeostasis by coordinating framework of anti-proliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on posttranslational modifications influencing its stability and activity, a growing number of evidences suggest that complex regulation of p53 expression occurs also at the mRNA level. This study explore structural determinants of long-range RNA-RNA interaction in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the eight nucleotide bulge motif plays a key structural role in base pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interaction in p53 mRNA. Nucleolin promotes base pairing of complementary sequences through the bulge motif since mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveal that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.

Ribonucleic acid (RNA) is a polymeric molecule that is fundamental to biological processes, with structure being more highly conserved than primary sequence and often key to its function. Advances in RNA structure characterization have resulted in an increase in the number of accurate secondary structures. The task of uncovering common RNA structural motifs with a collective function through structural comparison, providing a level of similarity, remains challenging and could be used to improve RNA secondary structure databases and discover new RNA families. In this work, we present a novel secondary structure alignment method, bpRNA-align. bpRNA-align is a customized global structural alignment method, utilizing an inverted (gap extend costs more than gap open) and context-specific affine gap penalty along with a structural, feature-specific substitution matrix to provide similarity scores. We evaluate our similarity scores in comparison to other methods, using affinity propagation clustering, applied to a benchmarking data set of known structure types. bpRNA-align shows improvement in clustering performance over a broad range of structure types.

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn, drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genome-wide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining relative m6A levels in mRNA populations from various sources based on enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

Analysis of tRNA modifications profile in several Archaea allowed to observe a novel modified uridine in the V-loop of several tRNAs from two species: Pyrococcus furiosus and Sulfolobus acidocaldarius (Wolff et al. 2020). Recently, Ohira et al. (Ohira et al. 2022) characterized 2'-phosphouridine (Up) at position 47 in tRNAs of thermophilic Sulfurisphaera tokodaii, as well as in several other archaea and thermophilic bacteria. From the presence of the gene arkI corresponding to the RNA kinase responsible for Up47 formation, they also concluded that Up47 should be present in tRNAs of other thermophilic Archaea. Re-analysis of our earlier data confirms that the unidentified residue in tRNAs of both P. furiosus and S. acidocaldarius is indeed 2'phosphouridine followed by m5C48. Moreover, we find this modification in several tRNAs of other Archaea and of the hyperthermophilic bacterium Aquifex aeolicus.