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Fragile X Syndrome (FXS) is characterized by intellectual impairment caused by CGG repeat expansion in the FMR1 gene. When repeats exceed 200, they induce DNA methylation of the promoter and the repeat region, resulting in transcriptional silencing of the FMR1 gene and the subsequent loss of FMRP protein. In the past decade or so, research has focused on the role of FMRP as an RNA-binding protein involved in translation inhibition in the brain in FXS model mice, particularly by slowing or stalling ribosome translocation on mRNA. More recent advances have shown that FMRP has a profound role in RNA splicing, at least in some cases by modulating the translation of splicing factor mRNAs. In a surprise, the human FMR1 gene is transcribed in most cases even with a full CGG expansion. However, much of the FMR1 that is produced is mis-spliced, which can be corrected by splice-switching antisense oligonucleotide (ASO) administration. Other recent findings suggest that inhibition of multiple kinases can demethylate the FMR1 gene and induce the formation of an R-loop in the CGG repeat region, leading to contraction of the repeat and FMRP restoration. These insights are paving the way for possible future therapeutic approaches for this disorder. We highlight the importance of FMRP restoration by ASO-mediated splice switching or CGG repeat modulation as key advances that may lead to successful treatments for FXS.

Mitochondrial tRNA (mt-tRNA) modifications play pivotal roles in decoding and sustaining tRNA stability, thereby enabling synthesis of essential respiratory complex proteins in mitochondria. Consequently, loss of human mt-tRNA modifications caused by mutations in the mitochondrial or nuclear genome can cause life-threatening mitochondrial diseases such as encephalopathy and cardiomyopathy. In this article, we first provide a comprehensive overview of the functions of mt-tRNA modifications, the responsible modification enzymes, and the diseases caused by loss of mt-tRNA modifications. We then discuss progress and potential strategies to treat these diseases, including taurine supplementation for MELAS patients, targeted deletion of mtDNA variants, and overexpression of modification-related proteins. Finally, we discuss factors that need to be overcome to cure 'mitochondrial tRNA modopathies'.

Recent studies revealed that the YTHDF family proteins bind preferentially to the N6-methyladenosine (m6A)-modified mRNA and regulate functions of these RNAs in different cell types. YTHDF2, the first identified m6A reader in mammals, has garnered significant attention because of its profound effect to regulate the m6A epitranscriptome in multiple biological processes. Here, we review current knowledge on the mechanisms by which YTHDF2 exerts its functions and discuss recent advances that underscore the multifaceted role of YTHDF2 in development, stem cell expansion and immune evasion. We also highlight potential therapeutic interventions targeting the m6A/YTHDF2 axis to improve the response to current antitumor therapies.

Viral quasispecies refers to the complex and dynamic mutant distributions (also termed mutant spectra, clouds or swarms) that arise as a result of high error rates during RNA genome replication. The mutant spectrum of individual RNA virus populations is modified by continuous generation of variant genomes, competition and interactions among them, environmental influences, bottleneck events, and bloc transmission of viral particles. Quasispecies dynamics provides a new perspective on how viruses adapt, evolve and cause disease, and sheds light on strategies to combat them. Molecular flexibility, together with ample opportunity of mutant cloud traffic in our global world, are key ingredients of viral disease emergences, as exemplified by the recent COVID-19 pandemic. In the present article we present a brief overview of the molecular basis of mutant swarm formation and dynamics, and how the latter relates to viral disease and epidemic spread. We outline future challenges derived of the highly diverse cellular world in which viruses are necessarily installed.

All RNAs exist in complexes (RNPs) with RNA-binding proteins (RBPs). Studies in my lab since the 1980s, identified, sequenced and characterized the major pre-mRNA- and mRNA-RBPs (hnRNPs/mRNPs), revealing RNA-binding domains and common features of numerous RBPs and their central roles in post-transcriptional gene regulation. The first links between RBPs and RNPs to diseases emerged serendipitously for fragile X syndrome, as its gene (FMR1) encoded RBP (FMRP), and spinal muscular atrophy (SMA), caused by deficits in survival motor neurons (SMN). Discoveries of the SMN complex and its unanticipated function in RNP assembly, essential for spliceosomal snRNPs biogenesis, advanced understanding of RNA biology and pathogenesis. I reflect on how these and other contributions (e.g., nucleo-cytoplasmic shuttling; telescripting) originated from curiosity-driven exploration and highly collaborative lab culture. The vast RNA and RBP assortments are beneficial, but increase complexity and chances of disorders, making the RNP sphere a rich source for future discoveries.

Cold-shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold-shock domain (CSD) that contains RNA-binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here, we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD, and the N-terminal portion with Lys4 of CspA, are important for determining their target specificities. Pull-down assays suggest that these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit a different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD-type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding the functional specialization of Csps.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.

During the chemical synthesis of the purine riboside, N7-regioisomer is kinetically formed, whereas N9-regioisomer is a thermodynamically formed product. We have studied the effect of substituting N9-regioisomer of guanosine with its N7-regioisomer (N7-guanosine, 7G) at a central position of several RNA duplexes. We found that this single substitution by 7G severely diminished their thermodynamic stabilities when 7G paired with C and U, but remarkably, led to a significant amount of stabilization in most of the duplexes when forming mismatches with G and A. The extent of stabilization was observed to be dependent on the sequence and orientation of neighboring base pairs of N7-guanosine. 1D and 2D NMR studies on the duplexes along with extensive molecular dynamics simulations revealed the conformational differences occurring due to the substitution of G by 7G, and it was observed that the thermodynamic results were largely explainable by considering the formation of stable noncanonical hydrogen bonding interactions, although other interactions such as stacking and electrostatic interactions could also play a role. These observations can have important applications in the design of RNA-based disease diagnostics and therapeutics.

Microexons (exons ≤30 nt) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in Caenorhabditis elegans Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A coregulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (∼30 nt) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by the skipping of a small "poison exon." Similar homeostatic cross-regulation is often observed across paralogous RNA-binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

Using a graph representation of RNA structures, we have studied the ensembles of secondary and tertiary graphs of two sets of RNA with Monte Carlo simulations. The first consisted of 91 target ribozyme and riboswitch sequences of moderate lengths (<150 nt) having a variety of secondary, H-type pseudoknots and kissing loop interactions. The second set consisted of 71 more diverse sequences across many RNA families. Using a simple empirical energy model for tertiary interactions and only sequence information for each target as input, the simulations examined how tertiary interactions impact the statistical mechanics of the fold ensembles. The results show that the graphs proliferate enormously when tertiary interactions are possible, producing an entropic driving force for the ensemble to access folds having tertiary structures even though they are overall energetically unfavorable in the energy model. For each of the targets in the two test sets, we assessed the quality of the model and the simulations by examining how well the simulated structures were able to predict the native fold, and compared the results to fold predictions from ViennaRNA. Our model generated good or excellent predictions in a large majority of the targets. Overall, this method was able to produce predictions of comparable quality to Vienna, but it outperformed Vienna for structures with H-type pseudoknots. The results suggest that while tertiary interactions are predicated on real-space contacts, their impacts on the folded structure of RNA can be captured by graph space information for sequences of moderate lengths, using a simple tertiary energy model for the loops, the base pairs, and base stacks.