RNA Biology

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

Transfer RNAs (tRNAs) are known for delivering amino acids to the growing polypeptide chain during translation. They can also influence gene expression, especially in times of nutrient starvation, through differential tRNA expression and modification. tRNAs have a highly consistent cloverleaf structure, but relatively few known regulatory elements govern this conserved structure despite the 20 different standard isotypes. This study examines gene enrichment patterns near tRNA in 1154 fungal genomes. Genes enriched in proteasome regulation, ion transport, and rRNA were found to be significantly closer to tRNAs than other pathways. These results were consistent across KEGG over-representation analysis (ORA), KEGG Gene Set Enrichment Analysis (GSEA), and Gene Ontology (GO) analysis. Proteasome, ion transport, and RNA are all important aspects of protein production and regulation, suggesting that genes required for the synthesis and quality control of proteins, including tRNAs, are located near each other. Protein regulation is an energetically expensive process, and local co-regulation could increase efficiency and stress impacts on proteins.

Ribosomal proteins, because of their RNA-binding capacity, may engage various cellular RNAs and fulfill nonribosomal roles. Previously, we and others described the intergenic regulation mediated by splicing of RPL22 paralogs in Saccharomyces cerevisiae. Here, we prepared a panel of RPL22A/B intronic mutants with respect to their RNAfold-predicted features and analyzed their properties. We tested the splicing and Rpl22-intron interaction using an intron-containing reporter and a three-hybrid yeast system, respectively. We found that the splicing of RPL22 introns can be inhibited by stabilizing a predicted stem as part of a particular type of conformation (I structure). Stabilizing the formation of an alternate stem (P structure) led to a permissive outcome of splicing. Intriguingly, the regulatory capacity of the main stem loop of the I structure was dependent on the rest of the intronic structure. Rpl22 enhanced splicing inhibition in WT and several of the mutants, which we interpret as stabilization of the I structure by protein binding. Mutagenesis identified both the main and alternative 5ss and additional stem loops as part of the regulatory mechanism. The inhibitory conformation of the intron did not prevent recognition of the 5ss and branch point, but rather stalled splicing at a later stage, before the first catalytic step. We concluded that the structural ensemble of the RPL22 pre-mRNA behaves as an allosteric switch that responds to [Rpl22].

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction, although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co-immunoprecipitation followed by phosphopeptide enrichment and high-resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co-immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insights into the posttranscriptional control of gene expression.

BackgroundSmall non-coding RNAs (sncRNAs) play central roles in post-transcriptional gene regulation. In addition to canonical microRNAs (miRNAs), fragments derived from vault RNAs (vtRNAs), called small vault RNAs (svtRNAs), have been reported in human cells. However, the absence of a standardized annotation framework has hindered their systematic detection, quantification, and comparison across small RNA sequencing (small RNA-seq) studies. MethodsWe developed an expression-based annotation strategy to identify svtRNAs from human small RNA-seq datasets. Using FlaiMapper followed by structure and expression-based filtering, we generated two annotation sets: a stringent "miRNA-like" set enriched in Argonaute-associated datasets, and (ii) a broader "Total" set derived from total small RNA-seq libraries under relaxed structural constraints. We explored the expression of the annotated svtRNAs across the different datasets analyzed: multiple normal and tumor-derived human cell lines, including Argonaute immunoprecipitation datasets. ResultsWe identified a repertoire of svtRNAs that are detected across independent datasets and, in several cases, reach abundance levels comparable to canonical miRNAs. Several highly abundant svtRNAs correspond to molecules with experimental validation from prior studies, supporting the robustness of our annotation strategy. Importantly, the same "dominant" (in terms of gene expression) svtRNAs emerged independently from Argonaute-associated and total small RNA datasets, supporting the idea of enzymatically consistent, reproducible svtRNA processing. We further identified svtRNAs derived from distinct vtRNA precursors that could share identical seed sequences, suggesting the possibility of svtRNA families with potential miRNA-like regulatory properties. We provide a standardized annotation that enables reproducible svtRNA quantification. ConclusionsOur study establishes a comprehensive expression-based annotation resource for human svtRNAs. By enabling their systematic detection and reproducible quantification, we show that svtRNAs appear to represent an abundant component of the human small RNA landscape.

1Circular RNAs have garnered considerable interest, as they have been implicated in numerous biological processes and diseases. Through their stability, they are often considered promising biomarker candidates or therapeutic targets. Due to the lack of a poly(A) tail, circRNAs are best detected in total RNA-seq data after depleting ribosomal RNA. However, we observe that the application of circRNA detection in the vastly more ubiquitous poly(A)-enriched RNA-seq data still occurs. In this study, we systematically compare the detection of circRNAs in two matched poly(A) and ribosomal RNA-depleted data sets. Our results indicate that the comparably few circRNAs detected in poly(A) data are likely false positives. In addition, we demonstrate that the quality of sample processing, as measured by the fraction of ribosomal reads, significantly affects the sensitivity of circRNA detection, leading to a bias in downstream analysis. Our findings establish best practices for circRNA research: total RNA sequencing with effective rRNA depletion is the preferred approach for accurate circRNA profiling, whereas poly(A)-enriched data are unsuitable for comprehensive detection. Employing multiple circRNA detection tools and prioritizing back-splice junctions identified by several algorithms enhances confidence in the selection of candidates. These recommendations, validated across diverse datasets and tissue types, provide generalizable principles for robust circRNA analysis. Key PointsO_LIRibosomal RNA contamination substantially impairs the accuracy of circRNA detection. This technical confounding factor has thus far received limited attention in the field. C_LIO_LITool agreement for circRNA calls is moderate in total RNA-seq but essentially absent in poly(A)-enriched RNA-seq data, underscoring the importance of using multiple tools for circRNA detection. C_LIO_LIBack-splice junctions detected in poly(A)-enriched RNA-seq data are predominantly tool-specific artifacts rather than genuine circRNAs, challenging the validity of circRNA identification in poly(A)-enriched datasets. C_LI

Viroids are small, circular non-coding RNAs that autonomously replicate in plants, exploiting host cellular machinery for replication and spread. Recent studies reveal that viroid-like agents can infect filamentous fungi, suggesting cross-kingdom interactions. In this study, we report the discovery and the characterization of TsvlRNA1 in Trichoderma spirale, a transmissible viroid-like RNA containing a hammerhead ribozyme in one polarity strand. Bioinformatic data, molecular validation, and reverse genetics experiments demonstrate that TsvlRNA1 is circular with an active ribozyme essential for replication. TsvlRNA1 replicates autonomously and transmits horizontally between Trichoderma species, eliciting 21-23 nt viroid-derived small RNAs consistent with RNA silencing targeting. The biocontrol capacity of Trichoderma against Rhizoctonia solani is variably modulated by TsvlRNA1, with effects ranging from positive to negative depending on host strain. In T. spirale, data suggests genotype-by-agent interactions influence antagonistic potential negatively. TsvlRNA1 transmission via horizontal routes is prevalent, and the viroid-like RNA fails to infect plant hosts experimentally. These results highlight so-far the underappreciated ecological and functional diversity of viroid-like agents in fungi, with implications for fungal biology, biocontrol, and genotype-phenotype relationships in eukaryotes. ImportanceSpecies of the fungal genus Trichoderma play a central role in sustainable agriculture by controlling fungal plant pathogens and supporting plant growth. For this reason, Trichoderma-based products represent a substantial share of the global market for microbial biofungicides. Viroids are the smallest known infectious agents, and their presence in filamentous fungi has only recently been discovered. Consequently, little is known about their biology, transmission, or interactions with fungal hosts. In this study, we describe TsvlRNA1, a viroid-like RNA associated with T. spirale, representing only the second viroid-like RNA to be biologically characterized in fungi. We show that TsvlRNA1 can influence the ability of Trichoderma to inhibit Rhizoctonia solani, a major plant pathogen, demonstrating its biological relevance. Unexpectedly, TsvlRNA1 can be transmitted between different Trichoderma species. This finding raises concerns about the possible transfer of genetic traits between fungi, including those related to fungicide resistance, with important implications for agricultural biocontrol. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/702247v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b6de6eorg.highwire.dtl.DTLVardef@c52141org.highwire.dtl.DTLVardef@a61fcorg.highwire.dtl.DTLVardef@1a6f0a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

MicroRNAs (miRNAs) play essential regulatory roles in controlling cell growth, proliferation, and differentiation in cancer. While functional studies have identified numerous oncogenic (oncomiRs) and tumor suppressor (TS) miRNAs, the structural features that differentiate these groups remain poorly understood. Here, we performed a comprehensive sequence analysis of 955 human pre-miRNA terminal loops (TLs), focusing on enrichment of single guanine (G) and GG dinucleotides. A quantitative G enrichment score was used to define 42 G-rich TL miRNAs and 17 G-free TL miRNAs as controls. Functional roles of these miRNAs were curated from 757 publications. The results show that G-rich TL miRNAs consistently display higher TS/oncomiR ratios than G-free TL miRNAs across most cancer types, with a significant enrichment observed in lung cancer (p = 0.023). Focusing on miR-139, a TS miRNA with a G-rich TL, integrative analysis of publicly available transcriptomic and proteomic data revealed its consistent downregulation across all stages of lung adenocarcinoma (LUAD), accompanied by reciprocal overexpression of its validated oncogenic target, CCNB1. These findings highlight the biological relevance of G-rich TL structures in miRNA-mediated tumor suppression in lung cancer, suggesting their consideration in future therapeutic strategies aimed at restoring vulnerable TS miRNAs.

Even though teleost fish and mammals share the same mitochondrial gene content and organization, the teleost mitochondrial transcriptome is still poorly understood. We characterized the mitochondrial transcriptome during zebrafish (Danio rerio) early development by long-read direct RNA sequencing. All heavy-strand specific mRNAs were found to carry 3 poly-A tails of approximately 50-60 residues, and the transcriptome profile was distinctive but practically invariant between stages. Three unusual transcripts were however noted. These included two mRNAs (COI and ND5 mRNAs), with significant 3 untranslated regions corresponding to antisense gene sequences, and a previously not described noncoding RNA named here lncOriL. The ND5 mRNA was found to carry one third of all detected m6A methylation sites in the zebrafish mitochondrial transcriptome. The 313 nt-long lncOriL transcript had an abundance comparable to that of ND5 mRNA and it mapped to mitochondrial genome region covering the origin of light strand replication and four flanking antisense tRNAs. A mitochondrial tRNA-derived fragment (tiRNA5-Asn), with a 35 nt perfect pairing-potential to lncOriL, was present at all stages. Additional analyses including adult zebrafish, scissortail (Rasbora rasbora), and monkfish (Lophius piscatorius) strongly corroborate the results of COI mRNA, ND5 mRNA, and lncOriL transcript prevalence among teleost fish. Surprisingly, our findings in zebrafish were further supported by mitochondrial transcriptome analyses in domestic pig (Sus scrofa) and human (Homo sapiens), including tiRNA5-Asn commonly present in human tissues, suggesting that lncOriL is ubiquitously expressed and regulated in vertebrates. Author SummaryMitochondria contain their own genome and produce essential RNAs needed for energy production. Although fish and mammals share the same mitochondrial gene organization, less is known about how mitochondrial RNAs are processed and regulated in teleost. Using Nanopore direct RNA sequencing, we examined mitochondrial RNAs during early zebrafish development and discovered three unusual transcripts that include extended non-coding regions. Two of these molecules, COI and ND5 mRNAs, carry long 3' untranslated regions formed by antisense gene sequences, suggesting previously unrecognized regulatory potential. We also identified lncOriL, a highly structured long noncoding RNA that spans the origin of light-strand replication and is abundant during development. Strikingly, the same RNA feature, including lncOriL and a matching tRNA-derived small RNA (tiRNA5-Asn), was found not only in zebrafish but also in human mitochondrial transcriptomes. These findings support conservation of regulatory mitochondrial RNAs across main groups of vertebrate species. Our work reveals a new layer of mitochondrial RNA regulation and expands the current understanding of how mitochondrial gene expression is controlled.

Long non-coding RNAs (lncRNAs) account for a major proportion of the transcriptional output in complex organismal genomes. Their emergence as auxiliary regulators of gene expression as well as their roles in metastasis and cancer progression has put them in the limelight. LncRNAs perform multitudes of functions and often moonlight as regulators, scaffolds and guides. Most lncRNAs are cell and tissue specific and can act as markers for diseases as well as targets for therapeutic interventions. LncRNAs are also known to make use of higher order structures such as G-quadruplexes (G4) to facilitate complex functions and interactions. THAP9-antisense1 (AS1) is a lncRNA coding gene (recently annotated by Ensembl) that codes for 12 lncRNA transcripts and has been implicated in many disease pathologies like gastric cancer, spontaneous neutrophil apoptosis, hepatocellular carcinoma, and the progression of oesophageal cancer. It is the antisense gene pair of the THAP9 gene ( a transposase derived gene) with which it shares a promoter. THAP9-AS1 has been reported to be dysregulated during stress and several cancers. However, the exact role of the lncRNA is not well understood. Bioinformatics driven strategies are used to identify putative quadruplex forming sequences (PQSs) within the lncRNA THAP9-AS1. The identified PQSs are further validated using biophysical, spectroscopic and molecular biology driven techniques. The importance of each G-tract in the formation of a particular RNA G-quadruplex (rG4) is studied via the investigation of several deletion mutants. The findings demonstrate the rG4 forming potential of the identified PQSs within THAP9-AS1.

Over the past decade, groundbreaking discoveries have cemented transfer RNAs (tRNAs) as versatile regulators of translation and cellular function. As tRNA research gains momentum, several high-throughput sequencing methods for quantitative analysis of tRNA isoacceptors in cells have emerged. However, the strong secondary structure and rich post-transcriptional modification of most tRNA molecules pose significant challenges for reverse transcriptases, thus hampering library preparation and introducing quantification biases. Current approaches rely on processive next generation reverse transcriptases (ngRTs), such as Induro (NEB) and uMRT (RNAConnect), to overcome these problems. Nevertheless, using these commercial enzymes comes with a relatively high cost per reaction. Here, we introduce a redesigned MarathonRT construct with added C-terminal chitin binding domain (CBD) and present a simple and robust one-step purification protocol for the in-house production of the redesigned and the original MarathonRT construct. Next, we set up an affordable colorimetry-based method for determining the specific activity of the enzyme. Our simplified method eliminates protein precipitation and yields over 26,000 enzymatic reactions per 0.5 L culture. Importantly, the in-house produced enzymes showed equal performance to established ngRTs Induro and uMRT in tRNA-seq workflow. In addition, we benchmark the use of rapid tRNA spin column-based extraction method to traditional gel-extraction using tRNA-seq and LC-MS. This improved workflow reduces the time and cost of tRNA-seq library preparation while providing an accessible MarathonRT purification protocol.

The majority of mammalian genes contains more than one functional cleavage and polyadenylation site, so that selection of alternative sites leads to different 3 ends of the mature transcripts. This mechanism can lead to the exclusion or retention of regulatory sequence elements, which affects post-transcriptional regulation of gene expression, such as RNA stability, localization or translation efficiency. We used 3 end sequencing to assess alternative polyadenylation during the response of macrophages to LPS, and observed a strong global shift towards proximal polyadenylation sites. This was accompanied by a decreased expression of cleavage and polyadenylation factors, including CPSF5, which is known to favor selection of distal polyadenylation sites. Upon depletion of CPSF5 in macrophage, we observed global transcript shortening, and an induction of TNF and other pro-inflammatory cytokines without LPS-stimulation. Analysis of RNA-seq data from monocytes of sepsis patients revealed that CPSF5 expression and alternative polyadenylation are also affected in vivo. Usage of distal polyadenylation sites showed a negative correlation with TNF mRNA expression in human monocytes. Our data suggest that transcript shortening mediated by CPSF5 repression contributes to the induction of pro-inflammatory genes.

BackgroundDDX53 (DEAD-box helicase 53, known also as CAGE) is an intronless gene on the X chromosome, which expression shows strong testis specificity. It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear. Previous reports identifying rare DDX53 variants in infertile men provided the rationale for investigating the role of DDX53 in the context of human spermatogenesis. By using the human seminoma cell line (TCam-2) as an in vitro male germline model, we aimed to investigate the function and molecular targets of DDX53. MethodsIn our study, we used transcriptomic and proteomic approaches (RNA sequencing (RNA-seq), enhanced crosslinking and immunoprecipitation (eCLIP), and Co-immunoprecipitation coupled with Mass Spectrometry (Co-IP-MS)) to investigate the role of DDX53 in the context of human spermatogenesis. By using modified TCam-2 cells to express either DDX53-FLAG or GFP-FLAG, we identified regulated genes, RNA targets, and potential protein interactors of DDX53. In addition, we employed Western Blot, RT-qPCR, immunostaining, and confocal microscopy to gain deeper insight into the DDX53 protein. ResultsOur RNA-seq and eCLIP data provide evidence that DDX53 regulates gene expression changes and directly interacts with a broad spectrum of RNA transcripts. Moreover, for the first time, we described RNAs and protein interactors of DDX53 in the context of spermatogenesis. Subcellular localization analysis by confocal microscopy indicated a predominantly cytoplasmic distribution of DDX53, with partial nuclear presence in TCam-2 cells. We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined. Additionally, we confirmed DDX53 presence in human testis using a specific, commercially available anti-DDX53 antibody. ConclusionsThis studys data indicate that DDX53 protein acts as a regulator of RNA metabolism in human cells. Collectively, we show that it participates in transcriptome regulation (including splicing) in male germ cells and exhibits transcriptome-wide RNA interactions, but its wider biological role remains to be clarified.

Conventional gene annotation pipelines classify eukaryotic genes into protein-coding and non-coding. Alternative splicing may generate non-coding transcript variants from protein-coding genes, that are expressed in tissue- or disease-specific manner. We and others have described the genes which transcribe both coding and non-coding transcripts as bifunctional genes. Here we present a genome-wide analyses of bifunctional genes and reannotate the genes in the human genome reference assembly into coding, non-coding and bifunctional. We identify over 4000 "bifunctional genes" in the human genome, constituting approximately 10% of the transcribed genes, and present evidence that these genes are conserved in evolution and their number correlate well with genome size and complexity. These genes are enriched in gene sets involved in vesicular transport, autophagy, RNA/DNA binding, glycosylation and splicing. By monitoring the expression of non-coding exons in long-read sequencing datasets and by quantitative RT-qPCR, we provide evidence for the expression of non-coding variants from bifunctional genes. The ncRNA transcripts from these genes might have similar or different roles from their cognate mRNA counterparts. They may act as miRNA sponges or harbour non-canonical open-reading frames that encode microproteins, while also competing for binding with RNA-binding proteins. We present evidence for establishing potential biological functions of bifunctional genes and summarise the findings in a searchable database. Further studies and functional characterization focused on this special group of genes may reveal interesting gene regulatory mechanisms relevant to physiology and pathology.

RNA structure plays a crucial role in diverse biological processes beyond the translation of genetic information. Therefore, the development of reliable methods for RNA structure prediction is essential for understanding RNA structure-related functions, however accurate and comprehensive RNA structure prediction remains challenging. Here, we focus on secondary structure prediction of transfer RNA (tRNA) using structure probing coupled with next-generation sequencing (tRNA Structure-seq). In silico prediction of Saccharomyces cerevisiae tRNA secondary structures achieves only 56.9% accuracy on average. Incorporation of dimethyl sulfate (DMS) probing data improve prediction accuracy to 87.4%, which is still not sufficient for practical tRNA structure prediction. To overcome this, we optimized the tRNA Structure-seq analysis pipeline by explicitly incorporating natural tRNA modifications detected in tRNA sequencing data and by refining pseudo-free energy parameters specifically optimized for tRNA structure prediction. Using this optimized pipeline, the average prediction accuracy is remarkably improved to 94%. Furthermore, analysis of multiple structural conformations predicted from DMS probing data indicates that S. cerevisiae tRNAs predominantly adopt the canonical cloverleaf secondary structure under in vivo conditions. Finally, we examined tRNA structures under mild stress conditions, including heat stress, osmotic stress, and antibiotic stress. These perturbations had minimal effects on in vivo tRNA secondary structure, demonstrating that S. cerevisiae tRNAs maintain structural stability under physiologically relevant stress conditions. In summary, our results establish an optimized tRNA Structure-seq analysis that enables highly accurate tRNA secondary structure prediction and reveals the intrinsic robustness of tRNA structures in living cells.

Accurate detection of RNA splice variants is often hindered when transcripts lack large distinguishable exonic regions, making conventional PCR strategies challenging. We developed a simple melting temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR method to enable variant-specific detection under these conditions. Uni-directional primers spanning exon-exon junctions were designed so that approximately each half anneals to adjacent exons. The Tm of each half-site was set >7{degrees}C below the annealing temperature, preventing stable binding to individual exons and enforcing junction-dependent amplification. The method was evaluated using HTRA1-AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1-AS1 comprises five variants, only one with a large distinguishable exon. Tm-guided EEJ primers robustly discriminated the remaining four variants. After optimization, amplification yielded sharp, single bands with minimal cross-reactivity. Compared with conventional designs, this approach reduced heteroduplex and heteroquadruplex formation, improving band clarity. Sanger sequencing confirmed junction specificity, and the method performed well in multiplex settings. Overall, Tm-guided EEJ RT-PCR is a cost-effective, high-resolution approach for detecting RNA variants lacking easily distinguishable exonic regions, readily compatible with standard RT-PCR and qPCR workflows.

Since the discovery of viral Internal Ribosome Entry Sites (IRESes), researchers have sought to find similar elements in mammalian host genes, termed "cellular IRESes". However, the plasmid systems used to measure cellular IRES activity are vulnerable to false positives due to promoter activity in candidate IRESes. Orthogonal methods are needed to validate putative IRESes while carefully avoiding artifacts known to cause false positives. Recently, Koch et al. proposed approaches for studying IRESes, primarily circular RNA-generating plasmids, and for validating mRNA transcripts using smFISH and qRT-PCR. Here, we demonstrate confounding variables and artifacts in each of these approaches that can lead to inappropriate conclusions about potential cellular IRES activity. We show the back-splicing circRNA plasmid creates linear mRNA artifacts associated with false-positive IRES signals. Using orthogonal, gold-standard assays validated with viral IRESes, we find putative cellular IRESes reported using the back-splicing plasmid have no IRES activity. Furthermore, we demonstrate that smFISH and qRT-PCR can misidentify nuclear non-coding RNAs as mRNAs and we validate a single molecule sequencing assay for identifying genuine mRNA 5 ends. Our work establishes reliable methods for robust transcript annotation and IRES studies that avoid documented artifacts arising from bicistronic and back-splicing circRNA plasmid reporters.

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

tRNA-derived fragments (tRFs) are relatively recently discovered class of small RNAs implicated in gene-regulatory processes in diverse biological contexts but there have been very few reports of a clear phenotypic role of these small RNAs in cancer progression. By analyzing small RNA-seq data from The Cancer Genome Atlas (TCGA), we found that high expression of three 3' tRFs (tRF-3a), tRF-3009a, tRF-3021a or tRF-3030a, is significantly associated with poor overall survival in low-grade glioma (LGG). In glioblastoma cells, tRF-3009a, tRF-3021a and tRF-3030a enhance cell invasion and migration but tRF-3021a was uniquely required for cell proliferation and suppression of apoptosis. Interestingly, tRF-3021a knockdown decreases global protein synthesis prior to and independent of apoptosis. These data indicate that tRF-3021a supports glioma cell survival and particularly protein synthesis while promoting cellular invasion and migration. Given its association with poor outcome in LGG patients, tRF-3021a represents a promising biomarker and potential therapeutic target in gliomas and these results provide a foundation for future studies to define its molecular interactors and downstream pathways controlling protein synthesis and apoptosis in cancer cells. ImplicationtRF-3021a promotes malignant glioma phenotypes, sustains global protein synthesis and prevents spontaneous apoptosis, motivating efforts to evaluate it as a biomarker and therapeutic target.