Translation reinitiation (REI) is one of the most important gene-specific regulatory mechanisms by which eukaryotic cells influence expression of main translons, for example during highly conserved integrated stress response (ISR). In S. cerevisiae, expression of the key stress response gene, GCN4, is controlled by an intricate interplay among four short upstream translons (uTranslons, formerly uORFs), resulting in high or low levels of REI at GCN4 depending on the growth conditions. Under nutrient rich conditions, GCN4 expression is repressed, but upon amino acid starvation, it is derepressed, despite of a general translational shut down. Capitalizing on our screening reporter system, we identified three new factors influencing efficiency of REI after translation of GCN4 uTranslons: Rai1p (an RNA quality control and processing factor), and Ssz1p and Zuo1p (members of the Ribosome Associated Complex [RAC]). Importantly, we showed that depletion of these factors deregulated derepression of Gcn4p synthesis under starvation. Furthermore, we found that similar to RAC, Rai1p associates with 40S subunits and actively translating ribosomes. We also explored interactomes of these proteins. Collectively, we present three previously unknown factors that co-regulate stress response to amino acid starvation in the budding yeast by unique mechanisms.

Accurate removal of intervening intronic sequences from pre-mRNA is required for proper eukaryotic gene expression. This process, termed pre-mRNA splicing, is carried out by a spliceosome, a dynamic RNA-protein macromolecular machinery. Proteomic studies have shown that many spliceosomal components and associated factors harbor protein arginine methylation, a type of post-translational modification. These findings raise the question of how arginine methylation influences the process or outcome of pre-mRNA splicing. Although targeted studies have provided important molecular insights into how this modification regulates splicing protein subcellular localization, abundance, RNA/protein interactions, and snRNP biogenesis, much remains unknown, especially given the large number of methylated proteins identified that have known roles in regulating pre-mRNA splicing. In this review, we summarize current knowledge of how protein arginine methylation contributes to pre-mRNA splicing and discuss how this modification may help tune the structural and functional plasticity of the spliceosome.

RNA-binding proteins (RBPs) regulate RNA metabolism at multiple levels and are essential mediators of post-transcriptional gene regulation in a wide range of physiological and pathological processes. The Rbm24/38 family is a conserved group of RRM-containing RBPs. In invertebrates, Rbm24/38 homologs are generally present as single-copy genes, whereas vertebrates possess two paralogs, Rbm24 and Rbm38, reflecting the progressive elaboration of posttranscriptional regulatory networks during evolution. Functional evidence for non-vertebrate Rbm24/38 homologs remains limited; the nematode protein SUP-12 has been primarily implicated in the regulation of pre-mRNA splicing during myogenesis. By contrast, vertebrate Rbm24 and Rbm38 exhibit broader spatial expression patterns across multiple tissues. Although their expression domains partially overlap, they also display clear gene- and species-specific differences. Functionally, vertebrate Rbm24 and Rbm38 contribute to multilayered post-transcriptional regulation through mechanisms such as alternative splicing, mRNA stability control, and alternative polyadenylation. In addition, regulatory features including isoform diversity, phase-separation potential, and post-translational modifications may further expand their functional versatility, enabling them to coordinate cell fate decisions and tissue homeostasis in distinct physiological contexts. In this review, we summarize recent advances in the evolution, structure, expression, molecular regulation, and biological functions of the Rbm24/38 family. We highlight its conserved features and functional diversification, as well as key unresolved questions regarding its evolution and specialization across metazoans.

RNA binding proteins (RBPs) interact with and tightly regulate the fate of messenger RNAs, but how RNA targets are recognized remains a challenging question. RBPs often contain multiple domains known to directly bind RNA, such as RNA recognition motifs (RRMs), as well as domains whose RNA binding capacity remains incompletely understood, e.g., low complexity domains (LCDs). Here, we dissect HNRNPR, an RBP with three RRMs and an arginine-glycine rich (RG-rich) LCD. We apply unbiased high-throughput biochemical approaches and identify critical RNA binding domains that confer specificity towards AU-rich RNA sequences. We show that not all RRMs contribute equally to binding and find that RRM3, along with a downstream C-terminal charged region, is required for RNA binding. HNRNPR also binds RNA G-quadruplexes (rG4s) and contains multiple rG4 binding sites including the C-terminal charged region within RRM3 and RG-rich regions within the LCD. We dissect rG4 specificity for the full length HNRNPR and LCD using a newly created RNA pool focused on rG4s, reveal that binding is dependent on RNA folding, and find specific rG4 features that enhance HNRNPR-rG4 interactions. Our work highlights the complex interplay of folded and disordered regions within RBPs as mediators of RNA binding.

Plasmodium falciparum, the primary cause of human malaria, relies on tightly coordinated gene-expression programs to adapt to host-derived stress despite possessing a limited repertoire of canonical transcription factors. Antisense long noncoding RNAs have emerged as important regulators of parasite biology, including virulence gene regulation and sexual commitment; however, their prevalence, origin, and broader functional significance remain poorly understood. Here, we demonstrate that antisense transcription is a widespread, reproducible, and regulated feature of the P. falciparum transcriptome rather than a byproduct of pervasive euchromatic transcription. Environmental stress, including febrile temperature exposure and artemisinin treatment, extensively remodelled antisense transcription, particularly at loci associated with virulence and stress adaptation, promoting widespread sense-antisense RNA duplex formation. Functional analyses of two stress-responsive chromatin regulators, PfGCN5 and PfHDAC1, identified as antisense-expressing loci, revealed that increased antisense expression elevated steady-state mRNA abundance while reducing cognate protein levels. Mechanistically, sense-antisense RNA duplex formation stabilized complementary transcripts but suppressed translation. Integrated transcriptomic, RNA-RNA duplex profiling, ribosome sequencing, and proteomic analyses further showed that duplex-enriched transcripts exhibit reduced ribosome occupancy and reduced protein abundance, accompanied by localized antisense enrichment near transcription end sites and altered ribosome distribution consistent with impaired translational engagement. Collectively, our findings identify an antisense RNA-ribosome regulatory axis that couples RNA duplex formation to adaptive translational control, providing a previously unrecognized mechanism underlying stress adaptation and post-transcriptional gene regulation in P. falciparum.

MicroRNA-mediated gene regulation occurs within spatially organized intracellular environments and is governed by dynamic molecular interactions that unfold across defined kinetic timescales. Although classical models emphasize sequence complementarity between microRNAs (miRNAs) and their target transcripts, growing evidence indicates that the outcomes of miRNA-target interactions vary widely across cellular contexts and cannot be explained solely by seed pairing or steady-state expression levels. These observations suggest that additional regulatory layers shape the efficiency and fate of miRNA-mediated silencing. In this Review, we examine how the spatial organization of the cell structures miRNA activity across distinct intracellular microenvironments, where the local availability of Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs), target RNAs and regulatory cofactors influences the probability that RISCs encounter and productively engage their targets. We further explore the temporal dimension of miRNA regulation, including Argonaute loading and turnover, the dwell time of RISC-target interactions, miRNA stability and target transcript lifetimes. Recent advances in quantitative and high-resolution approaches for studying RNA dynamics in living cells are beginning to reveal these regulatory processes directly. Together, these observations support a spatiotemporal kinetic model of miRNA regulation, in which intracellular organization and molecular dynamics jointly determine the probability and efficiency of gene silencing. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

RNA polymerase (RNAP) is the enzyme that produces RNA for an increasingly large variety of uses. The need for efficient industrial production of high quantities of mRNA was exemplified during the SARS-CoV-2 pandemic. While the RNAP from the T7 bacteriophage has been the gold standard for some time, it has certain limitations impeding efficient manufacturing of all RNA types, including production of long transcript self-amplifying RNA (saRNA). Here we present an alternative to T7 RNAP, identified from the Klebsiella bacteriophage 32 (KP32). We show KP32 RNAP to efficiently produce RNA of varying lengths, with a wide temperature, pH and salt tolerance range. We show KP32 RNAP to be capable of efficient production of an 11,551 nt self-amplifying RNA with its critical native initiating dinucleotide, AU. This represented a 9.3-fold improvement in production levels, relative to T7 RNAP.

The H/ACA ribonucleoprotein complex component dyskerin is essential for the biogenesis of H/ACA RNAs, including the human telomerase RNA (hTR). The N-terminal extension and α2' helix of dyskerin are hotspots for disease-associated mutations linked to X-linked dyskeratosis congenita (X-DC), a premature aging disorder. Some of these mutations disrupt dyskerin-hTR interactions, leading to hTR destabilization and reduced telomerase activity. Cryo-EM structures of human telomerase have shown that the N-terminal extension and α2' helix participate in dyskerin dimerization. However, biochemical evidence for dyskerin dimerization is still lacking, and it remains unclear whether mutations in these regions impair hTR binding by disrupting dimerization. Here, we provide the first biochemical evidence that dyskerin undergoes dimerization. We further demonstrate that dimerization is RNA independent and not abolished by disease mutations in the N-terminal extension or α2' helix. Instead, these mutations impair hTR binding. Our findings offer new mechanistic insight into how mutations in the dyskerin N-terminal extension and α2' helix contribute to the pathogenesis of X-DC.

The R2 retrotransposon family has been a leading model system for studies of site-specific non-long-terminal-repeat retrotransposon mobility. More recently, avian R2 systems have been harnessed to implement stable sequence supplementation of the human genome. Investigations of R2 retrotransposons range from detailed biochemical studies of purified components to elucidation of the cellular processes that support new retrotransposon insertions. In addition, molecular and genetic studies have examined the persistence and diversification of R2 retrotransposons across animals. In publications starting from early 2024, an R2-encoded protein has been used in native form, or after modification, to insert transgene sequences of choice into heterologous genomes lacking an endogenous R2. Many of these genome engineering studies were launched using a historically accumulated reporting of R2 sequences informative for understanding retrotransposon evolution but not intended as a catalogue of active R2 retrotransposons. Other groups approached R2 sequence identification by using or building consensus retrotransposon sequences from mostly short-read genome sequencing or genome assemblies. Here, we describe R2 sequence discovery, phylogeny, and annotation, and we consider their influence on R2 protein applications including gene addition to the human genome.

In 1986, a class of small, noncoding RNAs was discovered in association with an enormous, enigmatic ribonucleoprotein complex - the "vault" particle - and thus dubbed vault RNAs (vtRNAs). However, it's since been recognized that the vast majority (≥95%) of these noncoding vtRNA molecules are not associated with the mysterious vaults, raising questions about their potential independent function(s). Moreover, humans express four vtRNAs from two different loci, and debate has arisen about whether the vtRNA paralogs share any functional connection, and whether they should remain classified together. Herein, we report the expression patterns of the four VTRNA paralogs in a variety of human cell lines, including various single- and multi-gene VTRNA-knockout cell lines. Knockout of one or more of the three VTRNA1 genes leads to increased expression of vtRNA2-1, suggesting that their biological functions are related. Additionally, we interrogated the effects of vtRNA knockout on HEK293T cell growth, viability, and viral infection, and were unable to replicate previously reported associations. Collectively, our findings point to a potential functional connection between even the most distantly related human vtRNA paralogs, while reaffirming that their biological roles and mechanisms still require critical study.

Translating ribosomes can stall on mRNA for various reasons, including nuclease cleavage, arrest peptide sequences or ribosome collisions. In Escherichia coli, several ribosome rescue factors act to release ribosomes stalled at the 3' end of mRNA. Among these factors, ArfB can rescue stalled ribosomes without the help of other factors. ArfB consists of an N-terminal domain containing the catalytic GGQ motif, which mediates peptidyl-tRNA hydrolysis, and a C-terminal extension that functions as a sensor for recognizing stalled ribosomes. However, how these two regions coordinate to resolve ribosome stalling remains unclear. Here, using a reconstituted translation system, we found the functional importance of histidine residues in the N-terminal domain of ArfB. In particular, histidine at position 62 in E. coli ArfB was required for stop codon-independent fMet-tRNA hydrolysis, whereas its substitution did not affect affinity for the ribosome. Furthermore, directed hydroxyl radical probing revealed that H62A mutation did not significantly alter the overall positioning of either the N-terminal domain or the C-terminal extension of ArfB on the ribosome. These findings suggest that H62 contributes to ArfB function during a step following initial ribosome binding, thereby facilitating peptidyl-tRNA hydrolysis.

RNA splicing is the focus of a vast array of studies in biology, as it is involved in many normal cellular functions and related abnormalities. RNA-Seq remains the standard quantitative assay to interrogate splicing. The primary technical artifact of RNA-Seq library prep is the non-uniformity in coverage across transcripts. This undermines the accuracy of isoform-level quantification. This non-uniformity is present in both bulk and single-cell RNA-Seq and is purely artifactual as it is observed even when the sample contains only full-length transcripts. Therefore, understanding the source of this non-uniformity is of high importance. Here, we clarify the state of current knowledge and investigate the possible contributing factors. We demonstrate that no single factor can explain the phenomenon. Several approaches have previously been reported to improve uniformity, including reducing PCR ramp rates or cycle numbers, avoiding ribosomal depletion, using shorter fragment lengths, and using thermostable reverse transcriptases. We investigate all of these and demonstrate that none of these produce substantial improvements. We further investigate the possible sources of nonuniformity by assessing existing data sets with varying sample quality, PCR cycle number, reverse transcriptase, and technical or biological replicates. In addition, we generated novel data sets by varying PCR ramp rate, fragment length, and ribosomal depletion method. We demonstrate a connection of coverage with PCR that depends more on molecular abundance than PCR cycle number.

The discovery of tRNA as an adapter molecule established the foundation for understanding its essential roles in gene expression, stress responses, cellular homeostasis, and disease, including viral infections. Human tRNAs carry multiple chemical modifications that maintain their structure and ensure proper function. Since viruses depend entirely on host translation machinery, many of them can manipulate tRNA functioning at the structural level or the functional level. This review highlights how diverse viruses exploit tRNA epitranscriptomics, supported by recent advances in tRNA sequencing and profiling. Understanding the mechanisms by which viruses exploit the host tRNA landscape and the related molecules can identify novel antiviral targets, offering opportunities to interfere with viral protein synthesis and replication.

MicroRNA are pivotal regulators of organ and tissue homeostasis and development, and pathogenic mediators of disease. While antagonism of specific microRNA species in vivo is easily accomplished in experimental animal models using locked nucleic acid (LNA)-stabilized antimiRs, microRNA mimicry -that is, increasing the abundance of specific microRNA species- in experimental animals is more challenging. In this study, commercially available naked LNA-stabilized oligonucleotides designed as microRNA mimics were assessed for efficacy in newborn mouse pups, which frequently serve as models for normal human development, or neonatal and pediatric disease. Mimics targeting miR-497a-5p commercially marketed for in vivo or in vitro use were examined for distribution to five organs in developing mouse pups after parenteral administration. Mimics were most effectively delivered to the liver, and in limited instances also to the heart and kidneys, but could not be detected in the brain or lungs. A combination of bulk RNA-Seq transcriptomic screening and proteomic screening of liver homogenates revealed that mimics delivered to the liver were functional, either reducing the abundance of the Tff3 mRNA transcript, or the abundance of N4bp1 or Mt2 proteins, all of which are cognate targets of miR 497a-5p. Furthermore, the data presented here demonstrate that LNA-stabilized mimics for miR-497a-5p were short-lived in vivo, yet impacted the abundance of both primary and secondary targets of miR-497a-5p albeit with demonstrable hepatotoxicity. This study concludes that while microRNA mimicry using commercially-available naked LNA stabilized oligonucleotides is possible, there remains tremendous scope -and need- for refinement of this technology.

Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term PCanPIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization under near-physiological conditions (25 °C, 6 mM MgCl2), without the 55 °C heating step required by existing PIE systems. Kinetic analysis revealed that permutation site does not affect the observed splicing rate constant but does influence PCanPIE folding and therefore influences circularization efficiency. The P6b permutation yielded the highest circularization efficiency, with 95 % of the precursor splicing to produce circRNA. Optimization of spacer sequences flanking the circRNA payload eliminated interference from structured native exon sequences and enabled efficient circularization of RNAs up to 1,657 nt, including structured, repetitive, and naturally occurring sequences. Together, these results establish PCanPIE as a versatile and near-physiologically active addition to the group I intron PIE toolkit.