Project description:The roles of 3’-exoribonucleases and the exosome in trypanosome mRNA degradation; 30 min after actinomycin D +sinefungin, RNAi against CAf1, CNOT10, PAN2. These are really old data that hadn't been deposited.The datasets called RNA1, RNA2, RNA3 and RNA4 are almost certainly, from their location in the folder and from new alignment results, from the RRP45 RNAi.
Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
Project description:The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, the degradation of short lived non-coding RNAs and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degradation of distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before processing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild type cells are discarded before entering functional pathways, suggesting that kinetic competition with degradation proofreads the efficiency and accuracy of processing.
Project description:The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these exoribonucleases (Δpnp, Δrnr, Δrnb, and ΔrnbΔrnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of exoribonucleases in the transition between exponential and stationary phase.
Project description:Degradation of transcripts in mammalian nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, the Nuclear EXosome Targeting (NEXT) complex and the PolyA (pA) eXsome Targeting (PAXT) connection. However, how exact targeting is achieved remains enigmatic. Employing high-resolution 3’end sequencing of both steady state and newly produced pA+ and pA- RNA, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA- 3’ends often covering kb-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3’ends defined by canonical pA site usage. Irrespective this clear division, NEXT and PAXT act redundantly in two ways: i) Regional redundancy: The majority of exosome-targeted transcription units produce both NEXT- and PAXT-sensitive RNA isoforms; and ii) Isoform redundancy: The PAXT connection ensures the fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides for the efficient nuclear removal of superfluous RNA.
Project description:We report new insight of non-coding RNA degradation mediated by XRN exoribonucleases and FRY1 in Arabidopsis thaliana. We suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3’ extensions that arise from thousands of mRNA and miRNA precursor transcripts.
Project description:We report new insight of non-coding RNA degradation mediated by XRN exoribonucleases and FRY1 in Arabidopsis thaliana. We suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3’ extensions that arise from thousands of mRNA and miRNA precursor transcripts. Examination of genome-wide transcriptomes of Arabidopsis genotypes such as fry1-6, xrn3-3, xrn2-1xrn3-3, xrn2-1xrn4-6 and xrn3-3xrn4-6 using directional RNA-Seq method.