Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data from Drosophila S2 cells depleted of exosome subunits

ABSTRACT: The exosome complex and its' subunits are vital for proccessing and or degrading many cellular RNAs. We used microarrays to detail the global changes in mRNA levels in response to exosome subunit depletion. RNA was extracted from dsRNA treated S2 cells and hybridized using Affymetrix microarrays. We aimed to obtain a master set of mRNAs surveyed by exosme subunits in vivo.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Daniel kiss

PROVIDER: E-GEOD-17874 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits.

Kiss Daniel L DL Andrulis Erik D ED

RNA (New York, N.Y.) 20100225 4

The RNA processing exosome complex was originally defined as an evolutionarily conserved multisubunit complex of ribonucleases responsible for the processing and/or turnover of stable RNAs. The exosome complex is also involved in the surveillance of mRNAs in both the nucleus and the cytoplasm, including nonsense-mediated decay (NMD) targets. The detailed mechanisms for how individual exosome subunits participate in each of these RNA metabolic pathways remains unclear. Here, we use RNAi to deplet ...[more]

PMID: 20185544

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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.

Dataset Information

Expression data from Drosophila S2 cells depleted of exosome subunits

Publications

Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits.

Similar Datasets

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