RNA Biology

A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to "sponge" or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here we study whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ between its circular and linear forms. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.

Approximately a thousand microRNAs (miRNAs) are documented from human cells. A third appear to transit non-canonical pathways that typically bypass processing by Drosha, the dedicated nuclear miRNA producing enzyme. The largest class of non-canonical miRNAs are mirtrons which eschew Drosha to mature through spliceosome activity. While mirtrons are found in several configurations, the vast majority of human mirtron species are 5-tailed. For these mirtrons, a 3 splice site defines the 3 end of their hairpin precursor while a "tail" of variable length separates the 5 base of the hairpin from the nearest splice site. How this tail is removed is not understood. Here we examine sequence motifs in 5-tailed mirtrons and interactions with RNA turnover processes to characterize biogenesis processes. Through studying the high confidence 5-tailed mirtron, hsa-miR-5010, we identify RNaseP as necessary and sufficient for "severing" the 5 tail of this mirtron. Further, depletion of RNaseP activity globally decreased 5-tailed mirtron expression implicating this endoribonuclease in biogenesis of the entire class. Moreover, as 5-tailed mirtron biogenesis appears to be connected to tRNA processing we found a strong correlation between accumulation of tRNA fragments (tRFs) and 5-tailed mirtron abundance. This suggests that dysregulation of tRNA processing seen in cancers may also impact expression of the [~]400 5-tailed mirtrons encoded in the human genome. SUMMARYAbundant non-canonical human miRNAs referred to as tailed mirtrons are processed by RNaseP, which "severs" tail nucleotides to yield a precursor hairpin suitable for Dicer processing. Biogenesis of these miRNAs is correlated with tRFs, which are also products of RNaseP processing.

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y. Author summaryIn order to correctly regulate the level of RNAs, bacteria require their RNA to be continuously synthesised and degraded. However, even related bacterial species can have different sets of ribonucleases, each with their own target criteria. Here we explore which sequence elements of an RNA are important for being targeted by the endoribonuclese RNase Y in the two bacteria, Staphylococcus aureus and Bacillus subtilis. We specifically examine the RNase Y dependent cleavage of two transcripts that have homologs in both bacteria. We identify a short single-stranded regions immediately downstream of the cleavage position can be modified to change the cleavage efficiency up to 20-fold. We furthermore discover that a secondary structure a few nucleotides downstream of the cleavage is required for cleavage and that the positioning of the cleavage can be modulated by moving this structure.

The existence of translation regulation by RNA-induced silencing complexes (RISCs) composed from Argonaute proteins and micro-RNAs is well established, however the mechanisms underlying specific cellular and miRNA effects and the way in which specific complexes arise are not completely clear. Here we describe experiments with Renilla and Firefly luciferase reporter genes transfected on a PsiCheck2 plasmid into human cancer HCT116 or Me45 cells where only the Renilla gene contained or not sequences targeted by micro RNAs (miRNAs) in the 3UTR. The effects of targeting were miRNA-specific; miRNA-21 caused strong inhibition of translation whereas miRNA-24 or Let-7 caused no change or an increase in global reporter Renilla luciferase synthesis, and the mRNA-protein complexes formed by reporter transcripts in both cell types differed as shown by sucrose gradient sedimentation. In both cell types the presence of miRNA targets on Renilla transcripts affected expression of the co-transfected non-targeted Firefly luciferase, and Renilla and Firefly transcripts were found in the same sucrose gradient fractions. We also observed that specific anti-miRNA oligoribonucleotides influenced expression of the Firefly as well as of the Renilla gene, suggesting modulation of non-targeted transcript expression by miRNAs. Our results indicate the existence of interactions between miRNA-regulated and -unregulated transcripts and suggest that the use of the latter as a normalizers in experiments may be biased. We also discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.

Withdrawal StatementThe authors have withdrawn their manuscript owing to issues of cloning in the original pFmiR-MGAT3 sensor which make the data profiling miRNA regulation of this gene inaccurate. As a result, any miRNA identified should be disregarded and the data is invalid. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

The potential for miRNAs to regulate gene expression remains controversial. DROSHA initiates the biogenesis of miRNAs while Argonaute (AGO) and TNRC6 proteins form complexes with miRNAs that recognize RNA. Here we investigate the fate of miRNAs in the absence of critical RNAi protein factors. Knockout of DROSHA expression reduced levels of some miRNAs, but not others. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of miRNAs. Quantitative analysis indicates compensation to maintain the overall pool of AGO after knockout of AGO variants. Evaluation of miRNA binding to AGO proteins revealed that association between AGO and miRNAs was similar for AGO1 - 4. Contrary to the assumptions underlying many peer-reviewed reports, not all annotated miRNAs have equal potential as biological regulators. Cellular abundance, DROSHA dependence, and physical association with AGO must be considered when forming hypotheses related to their function. Our data prioritize sixty miRNAs - under two percent of the overall annotated miRNA repertoire - as being most likely to function as robust gene regulators. Our approach will facilitate identifying biologically active miRNAs.

The generation of tRNA halves and fragments (tsRNAs) has been associated with stressful growth conditions in eukaryotes, but reports on tsRNAs in bacteria are scarce. Here, we demonstrate that the presence of tsRNAs in Escherichia coli depends on active translation, and that they are found in the 30S ribosomal subunit and the ribosomal-free fraction, but not in the 50S subunits, 70S ribosomes or polysome fractions. However, upon dissociation into subunits at a low magnesium concentration, some of the tRNAs present in the monosomal and polysomal fractions are processed into tsRNAs. RNA-seq analyses tsRNA fractions revealed that all tRNA species in the cell were processed into fragment profiles that varied widely for each tRNA. The E. coli CP78 strain contains an unusually high concentration of tsRNAAsnGUU, but it is likely that only a fraction of this participates in translation. These tRNAs, along with others in the cell, were released from the ribosomes and processed into tsRNAs. The tRNAs in the ribosomal-free fraction appear to be cleaved by the same RNase that is active in ribosomes. We propose that tsRNAs are generated as an initial decay step for tRNAs remaining on ribosomes following translation arrest. However, tsRNAs may also have other functions.

MicroRNAs (miRNAs) are widely studied for their role in post-transcriptional gene regulation, often using exogenous overexpression systems to reveal their functions. However, such approaches may not accurately reflect endogenous miRNA activity due to the substantially higher expression levels achieved experimentally. To address this, we sought to determine the minimal endogenous expression threshold required for a miRNA to exert biologically significant effects. By comparing these experimentally determined expression thresholds with small RNA sequencing datasets comprising hundreds of cell lines and tens of thousands of tissue samples, we found that more than half of all annotated miRNAs are never expressed at levels sufficient to be biologically relevant. This calls into question the conclusions of thousands of studies reporting functions for these lowly expressed miRNAs, whose results are likely attributable to artificial overexpression rather than physiological activity. Our study highlights the need for more rigorous evaluation of miRNA functionality in their native context, and provides further support to arguments that the size of the functional human "microRNAome" is far smaller than some estimates of miRNA numbers based upon small RNA sequencing data.

Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei. While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3 end of the coding region, through the 3 tail, to the 5 start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.

BackgroundRNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. To date, most studies have focused on m6A, while other RNA modifications, including 5hmC, remain poorly characterised. The interplay between different RNA modifications that may occur in specific cellular contexts remains similarly unexplored. ResultsWe profiled m6A and 5hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. We observed that decreased expression of m6A and 5hmC writers, METTL3 and TET-enzymes respectively, facilitated monocyte-to-macrophage differentiation. Despite a global trend of m6A and 5hmC loss during macrophage differentiation, enrichment of m6A and/or 5hmC on specific categories of transcripts essential for macrophage differentiation positively correlated with their expression and translation. m6A and 5hmC mark and are associated with the expression of transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discovered the coexistence of m6A and 5hmC marking alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of transcripts with key roles in macrophage biology. In specific examples, RNA 5hmC controls the decay of transcripts independently of m6A. ConclusionsThis study: i) uncovers m6A, 5hmC and their writer enzymes as regulators of monocyte and macrophage gene expression programs and ii) provides a comprehensive dataset to interrogate the role of RNA modifications in a plastic system. Altogether, this work sheds light on the role of RNA modifications as central regulators of effector cells in innate immunity.

Following stress, tRNA is cleaved to generate tRNA halves (tiRNAs). These stress-induced small RNAs have been shown to regulate translation during stress. To date, angiogenin is considered the main enzyme that cleaves tRNA at its anti-codon site to generate 35 ~ 45 nucleotide long 5' and 3' tiRNA halves, however recent reports indicate the presence of angiogenin-independent cleavage. We previously observed tRNA cleavage pattern occurring away from the anti-codon site. To explore this non-canonical cleavage, we analyze tRNA phenotypical cleavage patterns in rat model of ischemia reperfusion and in two rat cell lines. In vivo mitochondrial tRNAs were prone to this non-canonical cleavage pattern. In vitro, however, both cytosolic and mitochondrial tRNAs could be cleaved non-canonically. We also evaluated the roles of angiogenin and its inhibitor, RNH1, in regulating tRNA cleavage during stress. Our results suggest that mitochondrial stress has an important regulatory role in angiogenin-mediated tRNA cleavage. Angiogenin does not appear to regulate the non-canonical cleavage pattern of tRNA, and RNH1 does not affect it as well. Finally, we verified our previous findings of the stress-specific role of Alkbh1 in regulating tRNA cleavage and showed a strong influence of stress type on Alkbh1-mediated tRNA cleavage and that Alkbh1 impacts non-canonical tRNA cleavage.

Tumor necrosis factor receptor superfamily 1A gene (TNFRSF1A) encodes the TNFR1 protein, a critical regulator of inflammation implicated in various diseases. Using ScanFold with the Integrative Genomics Viewer (IGV) GUI, we identified novel RNA structural elements within the TNFRSF1A gene. Focusing on the 3UTR, these structures were characterized using reporter assays and targeted DMS-MaPseq. We identified a structured region that may play a role in regulating TNFR1 translation and that was also found to associate with HuR, a key regulatory RNA-binding protein. These findings provide a framework for identifying and characterizing potential functional RNA structures in therapeutically relevant genes, suggesting a new layer of post-transcriptional regulation for TNFR1 expression.

Transfer ribonucleic acid (tRNA) are the smallest RNA in the translational triune and contain the greatest density of post-transcriptional modifications than any other RNA types in the cell. Due to the size of tRNA studying these modifications usually entails enzymatic digestion followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we report an advancement in Intact Mass Analysis for identification of tRNA through deconvolution of high resolution accurate mass spectrometry facilitated using a secondary alkylamine as an ion pairing reagent during reverse phase chromatography. We identify in isolated and total S. cerevisiae tRNA 3 CCA variations and show that most tRNA lack a 3 adenosine with the lesser abundant species having the expected CCA termini. We identify a previously unknown stable demethylated Wybutosine intermediate for tRNAPHE and identify low abundant contaminating tRNAs in an isolated tRNAPHE sample. Confirmation of identities was verified through traditional mass spectrometric nucleoside and mass mapping experiments.

It has been reported that a highly varying proportion (1% [~] 93%) of genes in various prokaryotes have antisense RNA (asRNA) transcription. However, the extent of the pervasiveness of asRNA transcription in the well-studied E. coli K12 strain has thus far been an issue of debate. Furthermore, very little is known about the expression patterns and functions of asRNAs under various conditions. To fill these gaps, we determined the transcriptomes and proteomes of E. coli K12 at multiple time points in five culture conditions using strand-specific RNA-seq, differential RNA-seq, and quantitative mass spectrometry methods. To reduce artifacts of possible transcriptional noise, we identified asRNA using stringent criteria with biological replicate verification and transcription start sites (TSSs) information included. We identified a total of 660 asRNAs, which were generally short and largely condition-dependently transcribed. We found that the proportions of the genes which had asRNA transcription highly depended on the culture conditions and time points. We classified the transcriptional activities of the genes in six transcriptional modes according to their relative levels of asRNA to mRNA. Many genes changed their transcriptional modes at different time points of the culture conditions, and such transitions can be described in a well-defined manner. Intriguingly, the protein levels and mRNA levels of genes in the sense-only/sense-dominant mode were moderately correlated, but the same was not true for genes in the balanced/antisense-dominant mode, in which asRNAs were at a comparable or higher level to mRNAs. These observations were further validated by western blot on candidate genes, where an increase in asRNA transcription diminished gene expression in one case and enhanced it in another. These results suggest that asRNAs may directly or indirectly regulate translation by forming duplexes with cognate mRNAs. Thus, asRNAs may play an important role in the bacteriums responses to environmental changes during growth and adaption to different environments. IMPORTANCEThe cis-antisense RNA (asRNA) is a type of understudied RNA molecules in prokaryotes, which is believed to be important in regulating gene expression. Our current understanding of asRNA is constrained by inconsistent reports about its identification and properties. These discrepancies are partially caused by a lack of sufficient samples, biological replicates, and culture conditions. This study aimed to overcome these disadvantages and identified 660 putative asRNAs using integrated information from strand-specific RNA-seq, differential RNA-seq, and mass spectrometry methods. In addition, we explored the relative expression between asRNAs and sense RNAs and investigated asRNA regulated transcriptional activity changes over different culture conditions and time points. Our work strongly suggests that asRNAs may play a crucial role in bacteriums responses to environmental changes during growth and adaption to different environments.

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].

Reactive oxygen species (ROS) are environmentally ubiquitous and known to have pervasive impacts on cellular homeostasis. RNA is vulnerable to oxidative chemical alterations from a variety of endogenous and exogenous sources. The most common chemical modification resulting from ROS exposure to RNA is 8-oxo-7,8-dihydroguanine (8-oxoG)--an oxidized form of the canonical guanine (G) nucleobase. While 8-oxoG modifications are known to impact mRNA processing, understanding the broader biological impact of 8-oxoG requires knowledge of how these modifications accumulate. In this work, we assessed the disparate enrichment of 8-oxoG modifications within RNAs in the E. coli transcriptome using an RNA Immunoprecipitation Sequencing technique with a high-affinity 8-oxoG antibody (8-oxoG-RIP-Seq). Our investigation of the RNA 8-oxoG enrichment landscape uncovered several intrinsic RNA characteristics that correlate with 8-oxoG enrichment. These findings suggest intrinsic characteristics of RNA, most notably relative abundance, CDS length, and G nucleotide composition, significantly influence RNA 8-oxoG accumulation. We harnessed these intrinsic characteristics to construct a simple multiple linear regression model that predicts RNA 8-oxoG accumulation, which we validated in E. coli. This model was subsequently applied to predict 8-oxoG enriched RNA species in four other bacterial species spanning a wide range of oxidative stress tolerances; these predictions suggest that 8-oxoG accumulation is largely species dependent, with limited overlap in RNAs and functional pathways that are more susceptible to elevated levels of 8-oxoG accumulation. Overall, these findings better inform understanding of RNA 8-oxoG patterns in bacteria and have broader impacts towards advancing knowledge of the connection between RNA oxidation and cellular homeostasis.

A large number of RNA modifications are known to affect processing and function of rRNA, tRNA and mRNA 1. The N4-acetylcytidine (ac4C) is the only known RNA acetylation event and is known to occur on rRNA, tRNA and mRNA 2,3. RNA modification by acetylation affects a number of biological processes, including translation and RNA stability 2. For a few RNA methyl modifications, a reversible nature has been demonstrated where specific writer proteins deposit the modification and eraser proteins can remove them by oxidative demethylation 4-6. The functionality of RNA modifications is often mediated by interaction with reader proteins that bind dependent on the presence of specific modifications 1. The NAT10 acetyltransferase has been firmly identified as the main writer of acetylation of cytidine ribonucleotides, but so far neither readers nor erasers of ac4C have been identified 2,3. Here we show, that ac4C is bound by the nucleolar protein NOP58 and deacetylated by SIRT7, for the first time demonstrating reversal by another mechanism than oxidative demethylation. NOP58 and SIRT7 are involved in snoRNA function and pre-ribosomal RNA processing 7-10, and using a NAT10 deficient cell line we can show that the reduction in ac4C levels affects both snoRNA sub-nuclear localization and pre-rRNA processing. SIRT7 can deacetylate RNA in vitro and endogenous levels of ac4C on snoRNA increase in a SIRT7 deficient cell line, supporting its endogenous function as an RNA deacetylase. In summary, we identify the first eraser and reader proteins of the RNA modification ac4C, respectively, and suggest an involvement of RNA acetylation in snoRNA function and pre-rRNA processing.

SINEUPs are antisense long non-coding RNAs that enhance translation of overlapping sense mRNAs through the activity of two domains: a SINEB2 sequence UP-regulating translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD). In this study, we demonstrate that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA is an Internal Ribosomal Entry Site (IRES) when acting in cis and that known viral and cellular IRES sequences can act as Effector Domain in synthetic SINEUPs. To identify natural IRES-containing, non-coding RNAs with SINEUP-like activity, we focused on circular RNAs showing that the non-coding circ5533, transcribed from the c-myc locus, enhances endogenous protein expression of its target PX Domain Containing Serine/Threonine Kinase Like (Pxk) by increasing mRNA association to polysomes. In summary, this study shows that natural and synthetic SINEUPs include linear and circular transcripts with an embedded IRES sequence as ED.