tRNA Precursors Research Articles

Divergent molecular assembly and catalytic mechanisms between bacterial and archaeal RNase P in pre-tRNA cleavage

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli (Escherichia coli) and Mja (Methanocaldococcus jannaschii) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P (EcoRPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms.

Assessing the diagnostic utility of tRNA-derived fragments as biomarkers of head and neck cancer

Roughly 54,000 individuals are diagnosed with head and neck cancers in the United States yearly. Transfer RNA-derived fragments (tRF) are the products of enzymatic cleavage of precursor tRNAs, and have been proposed for use as biomarkers of head and neck cancer. In this study, we aim to further analyze the utility that tRFs might provide as biomarkers of head and neck cancer. tRF read counts were obtained for 453 tumor and 44 adjacent normal tissue samples and used to construct a gradient boosting diagnostic model. Although we identified 129 tRFs that were significantly dysregulated between these samples, the model achieved a sensitivity of only 69 % and a specificity of 59 %. tRFs are thought to induce the degradation of mRNA transcripts containing a complementary “seed” region. Despite the above performances, we chose to explore this concept of translational regulation by analyzing these tRFs for inverse correlation to the expression of select oncogenes and tumor suppressor genes implicated in head and neck cancer. Among others, CysGCA 5′-half and LysCTT 3′-tRF were upregulated in the tumor samples, and corresponded to decreased expression of PIK3R1, AKT1, and CPEB3. These transcripts were further found to contain numerous significantly complementary sites at which tRF-mediated mRNA degradation might occur. Although these tRFs did appear to correlate to many of the oncogenic metrics analyzed, we believe that additional research is needed before they might be used to improve the diagnosis, treatment, and survival of patients with this disease.

The role of tRF-Val-CAC-010 in lung adenocarcinoma: implications for tumorigenesis and metastasis

ObjectiveTransfer RNA-derived fragments (tRFs) are short non-coding RNA (ncRNA) sequences, ranging from 14 to 30 nucleotides, produced through the precise cleavage of precursor and mature tRNAs. While tRFs have been implicated in various diseases, including cancer, their role in lung adenocarcinoma (LUAD) remains underexplored. This study aims to investigate the impact of tRF-Val-CAC-010, a specific tRF molecule, on the phenotype of LUAD cells and its role in tumorigenesis and progression in vivo.MethodsThe expression level of tRF-Val-CAC-010 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Specific inhibitors and mimics of tRF-Val-CAC-010 were synthesized for transient transfection. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8), while cell invasion and migration were evaluated through Transwell invasion and scratch assays. Flow cytometry was utilized to analyze cell cycle and apoptosis. The in vivo effects of tRF-Val-CAC-010 on tumor growth and metastasis were determined through tumor formation and metastasis imaging experiments in nude mice.ResultsThe expression level of tRF-Val-CAC-010 was upregulated in A549 and PC9 LUAD cells (P < 0.01). Suppression of tRF-Val-CAC-010 expression resulted in decreased proliferation of A549 and PC9 cells (P < 0.001), reduced invasion and migration of A549 (P < 0.05, P < 0.001) and PC9 cells (P < 0.05, P < 0.01), enhanced apoptosis in both A549 (P < 0.05) and PC9 cells (P < 0.05), and increased G2 phase cell cycle arrest in A549 cells (P < 0.05). In vivo, the tumor formation volume in the tRF-inhibitor group was significantly smaller than that in the model and tRF-NC groups (P < 0.05). The metastatic tumor flux value in the tRF-inhibitor group was also significantly lower than that in the model and tRF-NC groups (P < 0.05).ConclusionThis study demonstrates that tRF-Val-CAC-010 promotes proliferation, migration, and invasion of LUAD cells and induces apoptosis in vitro, however, its specific effects on the cell cycle require further elucidation. Additionally, tRF-Val-CAC-010 enhances tumor formation and metastasis in vivo. Therefore, tRF-Val-CAC-010 may serve as a novel diagnostic biomarker and potential therapeutic target for LUAD.

Exportin-5 binding precedes 5′- and 3′-end processing of tRNA precursors in Drosophila

Exportin5 (Exp5) is the major microRNA (miRNA) nuclear export factor and recognizes structural features of pre-miRNA hairpins, while it also exports other minihelix-containing RNAs. In Drosophila, Exp5 is suggested to play a major role in tRNA export because the gene encoding the canonical tRNA export factor Exportin-t is missing in its genome. To understand molecular functions of fly Exp5, we studied the Exp5/RNA interactome in the cell line S2R+ using the CLIP (crosslinking and immunoprecipitation) technology. The CLIP experiment captured known substrates such as tRNAs and miRNAs, and detected candidates of novel Exp5 substrates including various mRNAs and long non-coding RNAs (lncRNAs). Some mRNAs and lncRNAs enriched PAR-CLIP tags compared to their expression levels, suggesting selective binding of Exp5 to them. Intronless mRNAs tended to enrich PAR-CLIP tags, therefore we proposed that Exp5 might play a role in export of specific classes of mRNAs/lncRNAs. This result suggested that Drosophila Exp5 might have a wider variety of substrates than initially thought. Surprisingly, Exp5 CLIP reads often contained sequences corresponding to the flanking 5’-leaders and 3’-trailers of tRNAs, which were thought to be removed prior to nuclear export. In fact, we found pre-tRNAs before end-processing were present in the cytoplasm, supporting the idea that tRNA end-processing is a cytoplasmic event. In summary, our results provide a genome-wide list of Exp5 substrate candidates, and suggest that flies may lack a mechanism to distinguish pre-tRNAs with or without the flanking sequences.

Mitochondrial Genome-Encoded Long Noncoding RNA Cytochrome B (LncCytB) and Mitochondrial Ribonucleases in Diabetic Retinopathy.

Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5' ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1-LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients.

Transfer RNA‑derived small RNAs: A class of potential biomarkers in multiple cancers (Review).

Transfer (t)RNA-derived small RNAs (tsRNAs) are a class of novel non-coding small RNAs that are created via precise cleavage of tRNAs or tRNA precursors by different enzymes. tsRNAs are specific biological molecules that serve essential roles in cell proliferation, apoptosis, transcriptional regulation, post-transcriptional modification and translational regulation. Additionally, tsRNAs participate in the pathogenesis of several diseases, particularly in the development of malignant tumors. At present, the process of discovering and understanding the functions of tsRNAs is still in its early stages. The present review introduces the known biological functions and mechanisms of tsRNAs, and discusses the tsRNAs progression in several types of cancers as well as the possibility of tsRNAs becoming novel tumor biomarkers. Furthermore, tsRNAs may promote and hinder tumor formation according to different mechanisms and act as oncogenic or oncostatic molecules. Therefore, tsRNAs may be future potential tumor biomarkers or therapeutic targets.

Suppression of the Escherichia coli rnpA49 conditionally lethal phenotype by different compensatory mutations.

RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.

Unveiling the role of tRNA-derived small RNAs in MAPK signaling pathway: implications for cancer and beyond.

tRNA-derived small RNAs (tsRNAs) are novel small non-coding RNAs originating from mature or precursor tRNAs (pre-tRNA), typically spanning 14 to 30nt. The Mitogen-activated protein kinases (MAPK) pathway orchestrates cellular responses, influencing proliferation, differentiation, apoptosis, and transformation. tsRNAs influence the expression of the MAPK signaling pathway by targeting specific proteins within the pathway. Presently, four MAPK-linked tsRNAs have implications in gastric cancer (GC) and high-grade serous ovarian cancer (HGSOC). Notably, tRF-Glu-TTC-027 and tRF-Val-CAC-016 modulate MAPK-related protein expression, encompassing p38, Myc, ERK, CyclinD1, CyclinB, and c-Myc, hindering GC progression via MAPK pathway inhibition. Moreover, tRF-24-V29K9UV3IU and tRF-03357 remain unexplored in specific mechanisms. KEGG analysis posits varied tsRNAs in MAPK pathway modulation for diverse non-cancer maladies. Notably, high tRF-36-F900BY4D84KRIME and tRF-23-87R8WP9IY expression relates to varicose vein (VV) risk. Elevated tiRNA-Gly-GCC-001, tRF-Gly-GCC-012, tRF-Gly-GCC-013, and tRF-Gly-GCC-016 target spinal cord injury (SCI)-related brain-derived neurotrophic factor (BDNF), influencing MAPK expression. tRF-Gly-CCC-039 associates with diabetes foot sustained healing, while tRF-5014a inhibits autophagy-linked ATG5 in diabetic cardiomyopathy (DCM). Additionally, tsRNA-14783 influences keloid formation by regulating M2 macrophage polarization. Upregulation of tRF-Arg-ACG-007 and downregulation of tRF-Ser-GCT-008 are associated with diabetes. tsRNA-04002 alleviates Intervertebral disk degeneration (IDD) by targeting PRKCA. tsRNA-21109 alleviates Systemic lupus erythematosus (SLE) by inhibiting macrophage M1 polarization. The upregulated tiNA-Gly-GCC-002 and the downregulated tRF-Ala-AGC-010, tRF-Gln-CTG-005 and tRF-Leu-AAG-001 may be involved in the pathogenesis of Lupus nephritis (LN) by affecting the expression of MAPK pathway. Downregulation of tsRNA-1018, tsRNA-3045b, tsRNA-5021a and tsRNA-1020 affected the expression of MAPK pathway, thereby improving Acute lung injury (ALI). This review comprehensively dissects tsRNA roles in MAPK signaling across cancers and other diseases, illuminating a novel avenue for translational medical exploration.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

RNase P: Beyond Precursor tRNA Processing.

Ribonuclease P (RNase P) was first described in the 1970's as an endoribonuclease acting in the maturation of precursor transfer RNAs (tRNAs). More recent studies, however, have uncovered non-canonical roles for RNase P and its components. Here, we review the recent progress of its involvement in chromatin assembly, DNA damage response, and maintenance of genome stability with implications in tumorigenesis. The possibility of RNase P as a therapeutic target in cancer is also discussed.

Transcription complexes recruit a chaperone to perform cotranscriptional processing of tRNA

Leone etal.1 reveal that Pol III transcription complexes recruit a chaperone, HSP70, to execute cotranscriptional cleavage of precursor tRNA. HSP70 binds to the polymerase and translocates to nascent precursor tRNA and then tRNA. The last complex facilitates Pol III to engage in a new, efficient transcription cycle with another HSP70.

Oncogenic-tsRNA: A novel diagnostic and therapeutic molecule for cancer clinic.

tsRNA (tRNA-derived small RNA) is derived from mature tRNA or precursor tRNA (pre-tRNAs). It is lately found that tsRNA's aberrant expression is associated with tumor occurrence and development, it may be used a molecule of diagnosis and therapy. Based on the cleavage position of pre-tRNAs or mature tRNAs, tsRNAs are classified into two categories: tRNA-derived fragments (tRFs) and tRNA halves (also named tiRNAs or tRHs). tsRNAs display more stability within cells, tissues, and peripheral blood than other small non-coding RNAs (sncRNAs), and play a role of stable entities that function in various biological contexts, thus, they may serve as functional molecules in human disease. Recently, tsRNAs have been found in a large number of tumors including such as lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, and prostate cancer. Although the biological function of tsRNAs is still poorly understood, increasing evidences have indicated that tsRNAs have a great significance and potential in early tumor screening and diagnosis, therapeutic targets and application, and prognosis. In the present review, we mainly describe tsRNAs in tumors and their potential clinical value in early screening and diagnosis, therapeutic targets and application, and prognosis, it provides theoretical support and guidance for further revealing the therapeutic potential of tsRNAs in tumor.

A minimal RNA substrate with dual fluorescent probes enables rapid kinetics and provides insight into bacterial RNase P active site interactions.

Bacterial ribonuclease P (RNase P) is a tRNA processing endonuclease that occurs primarily as a ribonucleoprotein with a catalytic RNA subunit (P RNA). As one of the first ribozymes discovered, P RNA is a well-studied model system for understanding RNA catalysis and substrate recognition. Extensive structural and biochemical studies have revealed the structure of RNase P bound to precursor tRNA (ptRNA) and product tRNA. These studies also helped to define active site residues and propose the molecular interactions that are involved in substrate binding and catalysis. However, a detailed quantitative model of the reaction cycle that includes the structures of intermediates and the process of positioning active site metal ions for catalysis is lacking. To further this goal, we used a chemically modified minimal RNA duplex substrate (MD1) to establish a kinetic framework for measuring the functional effects of P RNA active site mutations. Substitution of U69, a critical nucleotide involved in active site Mg2+ binding, was found to reduce catalysis >500-fold as expected, but had no measurable effect on ptRNA binding kinetics. In contrast, the same U69 mutations had little effect on catalysis in Ca2+ compared to reactions containing native Mg2+ ions. CryoEM structures and SHAPE mapping suggested increased flexibility of U69 and adjacent nucleotides in Ca2+ compared to Mg2+. These results support a model in which slow catalysis in Ca2+ is due to inability to engage U69. These studies establish a set of experimental tools to analyze RNase P kinetics and mechanism and can be expanded to gain new insights into the assembly of the active RNase P-ptRNA complex.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

Depletion of tRNA CCA-adding enzyme in Mycobacterium tuberculosis leads to polyadenylation of transcripts and precursor tRNAs

In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase (rv3907c, PcnA) as a functional CCA-adding enzyme (CCAMtb) essential for viability of M. tuberculosis. The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCAMtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.

A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

DiffSegR: an RNA-seq data driven method for differential expression analysis using changepoint detection.

To fully understand gene regulation, it is necessary to have a thorough understanding of both the transcriptome and the enzymatic and RNA-binding activities that shape it. While many RNA-Seq-based tools have been developed to analyze the transcriptome, most only consider the abundance of sequencing reads along annotated patterns (such as genes). These annotations are typically incomplete, leading to errors in the differential expression analysis. To address this issue, we present DiffSegR - an R package that enables the discovery of transcriptome-wide expression differences between two biological conditions using RNA-Seq data. DiffSegR does not require prior annotation and uses a multiple changepoints detection algorithm to identify the boundaries of differentially expressed regions in the per-base log2 fold change. In a few minutes of computation, DiffSegR could rightfully predict the role of chloroplast ribonuclease Mini-III in rRNA maturation and chloroplast ribonuclease PNPase in (3'/5')-degradation of rRNA, mRNAand tRNA precursors as well as intron accumulation. We believe DiffSegR will benefit biologists working on transcriptomics as it allows access to information from a layer of the transcriptome overlooked by the classical differential expression analysis pipelines widely used today. DiffSegR is available at https://aliehrmann.github.io/DiffSegR/index.html.