Transcriptome-wide mapping of MARF1-bound mRNAs
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA630233 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11684917.fastq.gz | Fastqsanger.gz | |||
| SRR11684918.fastq.gz | Fastqsanger.gz | |||
| SRR11684919.fastq.gz | Fastqsanger.gz | |||
| SRR11684920.fastq.gz | Fastqsanger.gz |
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2014-07-03 | E-GEOD-51183 | biostudies-arrayexpress
Project description:The goal of this experiment was to identify Staufen1-bound mRNAs in prometaphase cells. Stau1 abundance fluctuates through the cell cycle: it is high in the G2 phase of the cell cycle and rapidly decreases as cells transit through mitosis. The importance of controlling Stau1 levels was underscored by the observation that its overexpression impairs mitosis as well as proliferation of transformed cell lines. As a major post-transcriptional regulator, Stau1 may exert its role(s) through the spatial and/or temporal regulation of its bound mRNAs. Therefore, to gather clues to support this possibility, we identified Stau1-bound mRNAs in prometaphase as a means to identify mRNAs that could be subjected to post-transcriptional modulation when Stau1 is partly degraded in mitosis.
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Project description:We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.
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Project description: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.
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