Unknown,Transcriptomics,Genomics,Proteomics

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A transcriptome-wide atlas of RNP compositions reveals diverse classes of mRNAs and lncRNAs

ABSTRACT: Aim: To determine how different classes of transcript (e.g. lncRNAs and mRNAs) are defined in the cell. Approach: We determined the transcriptome-wide targets of key RNA packaging, maturation, export and turnover factors. We used the CRAC technique, whereby RNA:protein interactions are fixed by UV irradiation of yeast cultures, and RNA:protein complexes obtained via a stringent multi-step purification. A mild RNase treatment fragments the bound RNAs, which are then used as templates for RT-PCR, prior to sequencing. This approach enabled us to compare the maturation and turnover pathways of mRNAs and lncRNAs. Results: Our data reveal that mRNA and lncRNA maturation pathways diverge prior to nuclear export, and 3' end processing emerges as a key step in determining transcript fate. Our analyses also reveal when and where the tested proteins bind to mRNAs, and thus offer much insight into the dynamic assembly of mRNPs. Analyses of reads with non-genome-encoded A-tails enabled us to distinguish proteins bound to stable poly(A) tails on full-length mRNAs, and to short oligo(A)4-5 tails on nuclear surveillance intermediates. This lead to the identification of a novel class of promoter-proximal ncRNAs, that we suggest arise from early termination within protein-coding genes. Identification of targets of RNA packaging, processing, export and turnover factors in wild-type cells; replicates included for some but not all samples; “BY” samples are negative controls, which use untagged strains

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Alex Tuck

PROVIDER: E-GEOD-46742 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A transcriptome-wide atlas of RNP composition reveals diverse classes of mRNAs and lncRNAs.

Tuck Alex Charles AC Tollervey David D

Cell 20130801 5

Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5' caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined d ...[more]

PMID: 23993093

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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.

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A transcriptome-wide atlas of RNP compositions reveals diverse classes of mRNAs and lncRNAs

Publications

A transcriptome-wide atlas of RNP composition reveals diverse classes of mRNAs and lncRNAs.

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