Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA624858 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11539118.fastq.gz | Fastqsanger.gz | |||
| SRR11539119.fastq.gz | Fastqsanger.gz | |||
| SRR11539120.fastq.gz | Fastqsanger.gz | |||
| SRR11539121.fastq.gz | Fastqsanger.gz | |||
| SRR11539122.fastq.gz | Fastqsanger.gz |
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Project description:In this study, we use small RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. Complemented and extended by the use of northern blots, our results demonstrate that tRNAs and Y RNAs in the vesicle-depleted extracellular compartment are released from cells as full-length precursors. Following their release, tRNAs and Y RNAs are processed by RNase 1 into distinct fragments. In addition, our findings show that standard sequencing methods employed to detect tRNA fragments leave many of such fragments undetected and that a combination of end healing, 3’ deacylation and RNA modification removal before library preparation can substantially improve detection of tRNA halves and reduce biases.
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Project description:Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our in vitro assay. These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.
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Project description:A comparative RNA-seq approach between R. sphaeroides 2.4.1 wild type and an RNase J deletion strain 2.4.1∆rnj revealed the accumulation of different mRNA fragments in the mutant. The structural characteristics of these RNA fragments suggest that RNase J is responsible for the decay of degradation intermediates that cannot serve as substrates for the 3´-to-5´ exoribonucleases.
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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.
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Project description:A comparative RNA-seq approach between R. sphaeroides 2.4.1 wild type and an RNase J deletion strain 2.4.1M-bM-^HM-^Frnj revealed the accumulation of different mRNA fragments in the mutant. The structural characteristics of these RNA fragments suggest that RNase J is responsible for the decay of degradation intermediates that cannot serve as substrates for the 3M-BM-4-to-5M-BM-4 exoribonucleases. Total RNA from exponentially grown cultures (OD660 0,4) of 2.4.1 wild type and 2.4.1M-bM-^HM-^Frnj has been isolated in triplicates using the hot phenol method and prepared for sequencing on a Illumina Genome Analyzer IIx machine.
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Project description:tRNA-derived small RNA including tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) plays significant roles in the various molecular mechanisms that underlie certain human diseases. of the generation of tRFs/tiRNAs and their potential roles during Dengue virus (DENV) infection is not yet known. Here, we have performed small RNA sequencing to identify the generation and alterations in tRF expression profiles of DENV infected Huh7 cells. Our results suggest that 733 tiRNAs/tRFs were found to be differentially expressed during DENV infection. Interestingly, 3’tRF population were found to be upregulated and i-tRF population were found to be downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the impact of differentially expressed tRFs on DENV pathogenesis. Our results suggest the significant involvement in transcriptional regulation via RNA polymerase II promoter and metabolic pathways. Overall, our study contributes significantly to our understanding of the roles played by tRFs in the complex dynamics of DENV infection.
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