High density mapping of 3' extremities of 5.8S rRNA maturation precursors in Arabidopsis exosome mutants
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE96946 | GEO | 2017/12/18
SECONDARY ACCESSION(S): PRJNA380303
REPOSITORIES: GEO
Similar Datasets
Project description:The Arabidopsis core exosome (Exo9) has a phosphorolytic activity due to the RRP41 subunit. The goal of this experiment was to determine the role of this intrinsic activity of Exo9 on the degradation of rRNA maturation by-products in Arabidopsis.
2017-12-18 | GSE96947 | GEO
Project description:we analyzed globally the effect of exosome processing on the nuclear pre-mRNA transcripts by inactivating either the RRP41 or DIS3 subunit of the exosome. Using SOLiD RNA sequencing technology, we report 30-120 million mapped cellular compartment specific reads per sample allowing the detection of unspliced pre-mRNAs. We show that RRP41 and DIS3 knockdowns stabilize an overlapping set of U12-type introns. Studying the global effect of the exosome (Rrp41 or Dis3 subunit) knockdown comparing to the control sample.
2014-05-26 | E-GEOD-52539 | biostudies-arrayexpress
Project description:The exosome complex plays a central role in RNA metabolism, and each of its core subunits is essential for viability in yeast However, comprehensive studies of exosome substrates and functional analyses of its subunits in multi.CELlular eukaryotes are lacking Here we show that, in sharp contrast to yeast and metazoan exosome complexes, individual subunits of the plant exosome core are functionally specialized Using whole-genome oligonucleotide tiling microarray analyses of csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41, we uncovered unexpected functional plasticity in the plant exosome core as well as generated a set of high-resolution genome-wide maps of Arabidopsis exosome targets These analyses provide evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants and reveal novel aspects of stable structural RNA metabolism Finally, numerous novel exosome substrates were discovered, including a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described This large collection of RNAs belong to a Òdeeply hiddenÓ layer of the transcriptome that is tightly repressed and can only be visualized upon inhibition of exosome activity These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes Keywords: Strand-specific gene expression analysis using whole-genome oligonucleotide tiling microarray analyses of three exosome subunits, using csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41.
2007-12-28 | GSE9317 | GEO
Project description:we analyzed globally the effect of exosome processing on the nuclear pre-mRNA transcripts by inactivating either the RRP41 or DIS3 subunit of the exosome. Using SOLiD RNA sequencing technology, we report 30-120 million mapped cellular compartment specific reads per sample allowing the detection of unspliced pre-mRNAs. We show that RRP41 and DIS3 knockdowns stabilize an overlapping set of U12-type introns.
2014-05-26 | GSE52539 | GEO
Project description:The RNA exosome complex functions in both the accurate processing and rapid degradation of many different classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. To address this we used UV crosslinking in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We performed comparative analyses on exosome complexes lacking the exonucleolytic activity of Rrp44, either carrying a mutation in the Rrp44 S1 RNA-binding domain, predicted to disfavor direct access to the Rrp44 exonuclease active site, or with multiple mutations in Rrp41, reported to block RNA passage through the central channel. Our analysis identified targets using preferentially channel-threading pathway such as mRNAs, 5S rRNA or scR1. Our results suggest as well that aborted tRNAs transcripts, released during transcription, would be rapidly degraded using this route. Alternatively, pre-tRNAs appears to access Rrp44 directly. Both routes seems to be involved for degradation or maturation of RNAPI transcripts. The Rrp41 mutations were found to block substrate passage to Rrp44 only for cytoplasmic mRNAs, apparently confirming the prediction of widening of the lumen in the nuclear, Rrp6-associated complex.
2017-03-20 | GSE90647 | GEO
Project description:High density mapping of 3' extremities of rRNA exosome substrates in Arabidopsis exosome mutants
| PRJNA380203 | ENA
Project description:RRP41 is one of the nine subunits constituting the core complex of the eukaryotic RNA exosome. Data presented here allowed a proteomic characterization of the interactome of wild type or mutated myc-tagged RRP41 proteins incorporated into the RNA exosome. Flowers from myc-tagged RRP41 expressing lines were used to affinity purify RNA exosomes, which were analyzed by nanoLC-MS/MS analysis. Non-transgenic Arabidopsis lines of Col-0 accession were used in mock-immunoprecipitation experiments as negative controls.
2018-01-02 | PXD007984 | Pride
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.
2012-09-25 | E-GEOD-40046 | biostudies-arrayexpress
Project description:Ribosomal RNAs (rRNAs) biogenesis are multistep processes requiring the activity of several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remains obscure. Here, using loss-of-function approaches in mouse embryonic stem cells and deep sequencing of rRNA intermediates, we investigate at nucleotide resolution the requirements of exonucleases known to be involved in 5.8S maturation, and explore the role of the Perlman syndrome-associated 3’-5’ exonuclease Dis3l2 in rRNA processing. We expand the repertoire of Dis3l2 targets to three 5.8S, 5S, and 28S rRNAs. We uncover a novel cytoplasmic intermediate that we name ‘7SB’ rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide level insight into the exonuclease requirements for mammalian rRNA processing.
2019-04-13 | GSE129734 | GEO
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex.
2012-09-25 | GSE40046 | GEO