Structural determinants for tRNA selective cleavage by RNase 2/EDN
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ABSTRACT:
PROVIDER: PRJNA1053393 | ENA |
REPOSITORIES: ENA
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Project description:tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of tRFs population. By comparing wild-type and knock-out macrophage cell lines our previous work (Lu L, et al. CMLS, 2022, 79: 209) revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening synthetic tRNAs, single-mutant variants and anticodon-loop DNA/RNA hairpins. By sequencing the tRFs products, we identified the cleavage selectivity by recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Knowledge of RNase 2 specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.
2024-05-29 | GSE250347 | GEO
Project description:To identify tRNA fragments regulated by angiogenin (ANG, Rnase 5), we sequenced 15-50nt small RNAs upon ANG overexpression and ANG knockout.
2019-10-01 | GSE130764 | GEO
Project description:Here we show using RNA-seq that cleavage by RNase E direct entry pervades in both the degradation and processing of RNA. We also give further evidence that direct entry is facilitated by cooperative interaction with segments in addition to the ones in which cleavage occurs. RNA seq profiles were compared between a temperature-sensitive mutant of rne and its congenic wild-type incubated at a non-permissive temperature. RNA seq profiles were also compared between samples before and after incubation with a 5'-sensing mutant of RNase E in vitro.
2014-06-10 | E-GEOD-58285 | biostudies-arrayexpress
Project description:Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. Moreover, mounting evidence supports a noncanonical role for tRNA cleavage products in the control of gene expression during diverse conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to altered tRNA transcription, suggesting that tRNA regulation may play an important role in mediating viral replication or the host response. To better understand how viral infection alters tRNA expression, we combined Ordered Two Template Relay (OTTR) with tRNA-specific bioinformatic software called tRAX to profile full-length tRNAs and fragmented tRNA-derived RNAs (tDRs) during infection with the model gammaherpesvirus, MHV68. We find that OTTR-tRAX is a powerful sequencing strategy for combined tRNA/tDR profiling, and reveal that MHV68 infection triggers pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tDRs. Fragments of virally-encoded tRNAs (virtRNAs), as well as virtRNA base modification signatures are also detectable during infection. We further dissected the biogenesis pathway of an MHV68-induced cleavage product from a pre-tRNA. Our data shows that pre-tDR-Tyr expression is dependent on the tRNA splicing factor, TSEN2, and that pre-tDR-Tyr expression is inhibited by the kinase, CLP1, which regulates tRNA splicing. Significantly, our findings suggest that CLP1 kinase is required for infectious gammaherpesvirus production, offering new insight into the importance of tRNA processing during viral infection.
2024-07-31 | GSE255627 | GEO
Project description:RNase P is the essential activity that performs the 5’ maturation of 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 understand their integration with gene expression pathways. 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 B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/B localize in the nucleus and find that their double knock out mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome wide tRNAseq approach, we observe that TRM1A/B are responsible for the m2,2G26 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/B mutants for specific tRNAs, in particular, tRNAs containing a m2,2G modification at position 26 that are strongly down-regulated in TRM1A/B mutants. Altogether, results indicate that the m2,2G adding enzymes TRM1A/B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNAs biogenesis.
2023-09-04 | GSE223876 | GEO
Project description:Identification of RNase III cleavage sites in Escherichia coli
| PRJEB26394 | ENA
Project description:In this study we generated 5'P libraries in wt and RNAse III mutant strains, grown to exponential and stationary phases. Libraries that retain short RNA fragments were also generated in both growth phases. After sequencing by Illumina NextSeq 500 system, reads were mapped to E. coli genome NC_000913.3. By comparing the read start counts per position in the wt and mutant strain libraries we identified the cleavage sites of RNase III.
2018-09-30 | E-MTAB-6704 | biostudies-arrayexpress
Project description:Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs, whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3’-fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post- transcriptional regulators.
2016-12-14 | GSE81869 | GEO
Project description:Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A–SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A–SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation.
2022-09-01 | GSE185827 | GEO
Project description:RNase P is essential to perform the 5’ maturation of 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 understand their integration with gene expression pathways. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 makes a complex with the tRNA methyl transferases TRM1A and B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/B localize in the nucleus and find that their double knock out mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome wide tRNAseq approach, we observe that TRM1A/B are responsible for the m2,2G26 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/B mutants for specific tRNAs, in particular, tRNAs containing a m2,2G modification at position 26 that are strongly down-regulated in TRM1A/B mutants. Altogether, results indicate that the m2,2G adding enzymes TRM1A/B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNAs biogenesis.
2023-10-20 | PXD040022 | Pride