Measuring RNA folding is particularly challenging for tRNAs, where extensive structure and posttranscriptional modifications complicate traditional structure probing and reverse-transcriptase-based assays. Since tRNA folding is a prerequisite for aminoacylation, quantifying tRNA aminoacylation is often used as an indirect, though functionally relevant, measure of tRNA folding. Periodate oxidation and beta-elimination, resulting in the selective loss of the 3' terminal nucleotide of deacylated tRNAs, is an amino-acid-agnostic chemistry that enables differentiation of aminoacylated and deacylated tRNAs when coupled to high-resolution northern blotting or RNA sequencing. Quantification of tRNA aminoacylation has been used to demonstrate RNA chaperone activity for RNA-binding proteins that promote tRNA folding to increase the fraction of aminoacylated tRNAs, underscoring the utility of this assay in interrogating RNA chaperone-assisted tRNA folding and function.

La proteins have well-established roles in the maturation of RNA polymerase III transcripts, including pre-tRNAs. In addition to protecting the 3' end of pre-tRNAs from exonuclease digestion, La proteins also promote the native fold of the pre-tRNA using RNA chaperone activity. tRNA-mediated suppression in the fission yeast S. pombe has been an invaluable tool in determining the mechanistic basis by which La proteins promote the maturation of defective pre-tRNAs that benefit from RNA chaperone activity. More recently, tRNA-mediated suppression has been adapted to test for RNA chaperone function in the La-related proteins and in the promoting of tRNA function by tRNA modification enzymes. Thus tRNA-mediated suppression can be a useful assay for the investigation of various proteins hypothesized to promote tRNA folding through RNA chaperone related activities.

Small nucleolar RNAs (snoRNAs), a group of noncoding RNAs present amongst all eukaryotes, are most extensively characterized for their regulation of ribosome biogenesis and splicing. Despite their central roles, current snoRNA annotations remain incomplete. Several eukaryote genome annotations contain few or no snoRNAs, and none distinguish expressed snoRNAs from their pseudogenes-a recently characterized snoRNA subclass with distinct features and expression levels. To address this, we developed SnoBIRD, a BERT-based C/D box snoRNA predictor trained on snoRNAs spanning all eukaryote kingdoms. We show that SnoBIRD outperforms existing tools and is the only predictor capable of identifying snoRNA pseudogenes using biologically relevant signal. Applied on the fission yeast and human genomes, we demonstrate that only SnoBIRD scales well with genome size in terms of runtime, and we identify and experimentally validate several new SnoBIRD-predicted C/D box snoRNAs. By running SnoBIRD on multiple eukaryote genomes, we identify hundreds of novel snoRNA candidates and highlight SnoBIRD's usefulness to determine the evolutionary paths of snoRNAs distributed across different species. Overall, SnoBIRD represents a user-friendly and efficient tool for reliably predicting C/D box snoRNAs and their pseudogenes across any eukaryote genome.

La and La-related proteins (LARPs) are conserved RNA-binding proteins that share a characteristic La motif (LaM) and have important functions in RNA metabolism. Members of the LARP1 family bind a cohort of mRNAs encoding factors involved in the process of mRNA translation, including ribosomal protein mRNAs (RP mRNAs). These mRNAs can contain a sequence of 5-15 pyrimidines in their 5'UTRs, immediately following the m7G cap, and are named 5' terminal oligopyrimidine (5'TOP) mRNAs. The DM15 domain of human LARP1 has been suggested to specifically recognize this motif, thereby affecting 5'TOP mRNA translation and stability. However, the specific function of LARP1 in this context remains unclear. Intriguingly, the 5'TOP motif is not found in RP mRNAs in C. elegans and yeast, while LARP1 orthologs in some systems lack the characteristic DM15 domain, suggesting that essential functions of LARP1 family members may precede the emergence of the 5'TOP motif and the DM15. In this work, we review studies in humans and several model organisms where we draw parallels between reported RNA binding modes and functions of different LARP1 orthologs. We further present common themes and areas for further investigation. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Viruses strongly rely on the host’s translational machinery to produce viral proteins required for replication. However, it is unknown how viruses that do not globally inhibit cap-dependent translation compete with abundant host transcripts for ribosomes. Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5’-cap-dependent translation inhibition. Respiratory syncytial virus (RSV) encodes mRNAs mimicking 5’-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. RSV-infected HEp-2 and A549 cells were analyzed to determine translation levels using western blotting, indirect immunofluorescent staining and polysome profiling. Transcriptome-wide translation efficiencies of virus-infected cells were compared against mock-infected cells using high-throughput sequencing of poly(A)-tail enriched total mRNA and transcripts associated with heavy polysomes. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. High-throughput sequencing revealed that virus-resembling, AU-rich host transcripts become more efficient at ribosome recruitment. Using a previously published dataset, we observe similar trends in another negative-sense single-stranded RNA virus, vesicular stomatitis virus (VSV). These findings revealed that RSV changes the translational landscape by enhancing translation of virus-resembling AU-rich host transcripts rather than inhibiting host translation.

The enzyme complex KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) installs the universally conserved and essential N6-threonylcarbamoyl adenosine modification (t6A) on ANN-decoding tRNAs in eukaryotes and in archaea. KEOPS consists of Cgi121, Kae1, Pcc1, Gon7 and the atypical kinase/ATPase Bud32. Except Gon7, all KEOPS subunits are needed for tRNA modification, and in humans, mutations in all five genes underlie the lethal genetic disease Galloway Mowat Syndrome (GAMOS). Kae1 catalyzes the modification of tRNA, but the specific contributions of Bud32 and the other subunits are less clear. Here we solved cryogenic electron microscopy structures of KEOPS with and without a tRNA substrate. We uncover distinct flexibility of KEOPS-bound tRNA revealing a conformational change that may enable its modification by Kae1. We further identified a contact between a flipped-out base of the tRNA and an arginine residue in C-terminal tail of Bud32 that correlates with the conformational change in the tRNA. We also uncover contact surfaces within the KEOPS-tRNA holo-enzyme substrate complex that are required for Bud32 ATPase regulation and t6A modification activity. Our findings uncover inner workings of KEOPS including a basis for substrate specificity and why Kae1 depends on all other subunits.

The field of RNA research has provided profound insights into the basic mechanisms modulating the function and adaption of biological systems. RNA has also been at the center stage in the development of transformative biotechnological and medical applications, perhaps most notably was the advent of mRNA vaccines that were critical in helping humanity through the Covid19 pandemic. Unbeknownst to many, Canada boasts a diverse community of RNA scientists, spanning multiple disciplines and locations, whose cutting-edge research has established a rich track record of contributions across various aspects of RNA science over many decades. Through this position paper, we seek to highlight key contributions made by Canadian investigators to the RNA field, via both thematic and historical viewpoints. We also discuss initiatives underway to organize and enhance the impact of the Canadian RNA research community, particularly focusing on the creation of the not-for-profit organization RNA Canada ARN. Considering the strategic importance of RNA research in biology and medicine, and its considerable potential to help address major challenges facing humanity, sustained support of this sector will be critical to help Canadian scientists play key roles in the ongoing RNA revolution and the many benefits this could bring about to Canada.

Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5'-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5'-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

Protein -nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein -DNA or protein -RNA interactions in vitro . We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al. 1

Erythropoietin (EPO) suppresses drug-induced apoptosis in EPO-receptor-positive leukemia cells and allows cells to persist after drug treatment by promoting cellular senescence. Importantly a small proportion of senescent cells can re-enter the cell cycle and resume proliferation after drug treatment, resulting in disease recurrence/persistence. Using a single-cell assay to track individual cells that exit a drug-induced senescence-like state, we show that cells exhibit asynchronous exit from a senescent-like state, and display different rates of proliferation. Escaped cells retain sensitivity to drug treatment, but display inter-clonal variability. We also find heterogeneity in gene expression with some of the escaped clones retaining senescence-associated gene expression. Senescent leukemia cells exhibit changes in gene expression that affect metabolism and senescence-associated secretory phenotype (SASP)-related genes. Herein, we generate a senescence gene signature and show that this signature is a prognostic marker of worse overall survival in AML and multiple other cancers. A portion of senescent leukemia cells depend on lysosome activity; chloroquine, an inhibitor of lysosome activity, promotes senolysis of some senescent leukemia cells. Our study indicates that the serious risks associated with the use of erythropoietin-stimulating agents (ESAs) in anemic cancer patients may be attributed to their ability to promote drug-tolerant cancer cells through the senescence program.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5’ γ-monomethyl phosphate cap by members of the Bin3/MePCE family. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 also interact in a U6-containing snRNP. We demonstrate that Bmc1 and Pof8 promote 2’-O-methylation of U6 and identify and characterize a non-canonical snoRNA that guides this methylation. Further, we show that fission yeast strains deleted of Bmc1 or Pof8 show altered U6 snRNP assembly patterns, supporting a more general role for these factors in guiding noncoding RNP assembly beyond the telomerase RNP. These results are thus consistent with a novel role for Bmc1/MePCE family members in stimulating U6 post-transcriptional modifications.

tRNAs undergo an extensive maturation process involving posttranscriptional modifications often associated with tRNA structural stability and promoting the native fold. Impaired posttranscriptional modification has been linked to human disease, likely through defects in translation, mitochondrial function, and increased susceptibility to degradation by various tRNA decay pathways. More recently, evidence has emerged that bacterial tRNA modification enzymes can act as tRNA chaperones to guide tRNA folding in a manner independent from catalytic activity. Here, we provide evidence that the fission yeast tRNA methyltransferase Trm1, which dimethylates nuclear- and mitochondrial-encoded tRNAs at G26, can also promote tRNA functionality in the absence of catalysis. We show that WT and catalytic-dead Trm1 are active in an in vivo tRNA-mediated suppression assay and possess RNA strand annealing and dissociation activity in vitro, similar to previously characterized RNA chaperones. Trm1 and the RNA chaperone La have previously been proposed to function synergistically in promoting tRNA maturation, yet we surprisingly demonstrate that La binding to nascent pre-tRNAs decreases Trm1 tRNA dimethylation in vivo and in vitro. Collectively, these results support the hypothesis for tRNA modification enzymes that combine catalytic and noncatalytic activities to promote tRNA maturation, as well as expand our understanding of how La function can influence tRNA modification.

Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the trans-lational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the trans-lation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.

The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bmc1/Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bmc1 associates with the telomerase holoenzyme and U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bmc1 with telomerase is independent of its methyltransferase activity, but rather that Bmc1 functions in telomerase holoenzyme assembly by promoting TER1 accumulation and Pof8 recruitment to TER1. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation. There exists a remarkable evolutionary breadth of telomerase holoenzyme components among eukaryotes. Here the authors report that the fission yeast methyl phosphate capping enzyme Bin3/Bmc1 regulates telomerase activity by promoting holoenzyme formation.

Nascent pre-tRNAs are transcribed by RNA polymerase III and immediately bound by La proteins on the UUU-3'OH sequence, using a tandem arrangement of the La motif and an adjacent RNA recognition motif-1 (RRM1), resulting in protection from 3'-exonucleases and promotion of pre-tRNA folding. The Tetrahymena thermophila protein Mlp1 has been previously classified as a genuine La protein, despite the predicted absence of the RRM1. We find that Mlp1 functions as a La protein through binding of pre-tRNAs, and affects pre-tRNA processing in Tetrahymena thermophila and when expressed in fission yeast. However, unlike in other examined eukaryotes, depletion of Mlp1 results in 3'-trailer stabilization. The 3'-trailers in Tetrahymena thermophila are uniquely short relative to other examined eukaryotes, and 5'-leaders have evolved to disfavour pre-tRNA leader/trailer pairing. Our data indicate that this variant Mlp1 architecture is linked to an altered, novel mechanism of tRNA processing in Tetrahymena thermophila.

tRNAs undergo an extensive maturation process including posttranscriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding of misfolded substrates into functional conformations. The best characterized tRNA chaperone is the La protein, which interacts with nascent RNA polymerase III transcripts to promote folding and offers protection from exonucleases. More recently, certain tRNA modification enzymes have also been demonstrated to possess tRNA folding activity distinct from their catalytic activity, suggesting that they may act as tRNA chaperones. In this review, we will discuss pioneering studies relating posttranscriptional modification to tRNA stability and decay pathways, present recent advances into the mechanism by which the RNA chaperone La assists pre-tRNA maturation, and summarize emerging research directions aimed at characterizing modification enzymes as tRNA chaperones. Together, these findings shed light on the importance of tRNA folding and how tRNA chaperones, in particular, increase the fraction of nascent pre-tRNAs that adopt a folded, functional conformation.

The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bin3 associates with the telomerase and the U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bin3 with telomerase is independent of its methyltransferase activity, but rather that Bin3 negatively regulates TER1 levels and telomere length. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation. ### Competing Interest Statement The authors have declared no competing interest.

This special issue of RNA Biology is devoted to the La-related family of proteins (LARPs) and celebrates the 10th Anniversary of The LARP Society (colloquially known as LARPSOC). LARP Society, not ...