Extensive structural differences of closely related 3’ mRNA isoforms: links to Pab1 binding and mRNA stability
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ABSTRACT:
PROVIDER: PRJNA378410 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5319140_1.fastq.gz | Fastqsanger.gz | |||
| SRR5319140_2.fastq.gz | Fastqsanger.gz | |||
| SRR5319150_1.fastq.gz | Fastqsanger.gz | |||
| SRR5319150_2.fastq.gz | Fastqsanger.gz | |||
| SRR5319160.fastq.gz | Fastqsanger.gz |
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Project description:Alternative polyadenylation generates numerous 3' mRNA isoforms per gene; regulation of this process is critical in development and impaired in cancer. Individual isoforms from the same gene can vary greatly in biological properties such as translational efficiency, localization, and half-life. Even closely related isoforms - including those differing by a single nt - can have different biological properties, but the underlying mechanisms are unknown. Here we show that many highly similar yeast isoforms unexpectedly exhibit extensive structural variation and differential Pab1 binding, and that these physical properties serve as a good predictor of relative isoform stability. We developed DREADS, a dimethyl sulphate (DMS)-based technique, to obtain the first transcriptome-scale structural description of individual 3' mRNA isoforms in vivo. Strikingly, near-identical yeast mRNA isoforms arising from the same gene can possess dramatically different DMS modification profiles over hundreds of nt upstream of the poly(A) tail. DREADS analyses of the same mRNAs refolded in vitro or in different species indicate that structural differences in vivo are often due to trans- acting factors. CLIP-READS, a technique we developed to measure isoform-specific binding, reveals that differences in Poly(A) binding protein (Pab1) binding to 3' ends correlate with the extent of structural variation for closely-spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-stranded character of the poly(A) tail, and low Pab1 binding is significantly associated with mRNA stability. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, and are indicative of biological function.
2018-10-11 | GSE95788 | GEO
Project description:Alternative polyadenylation generates numerous 3' mRNA isoforms that can vary in biological properties, such as stability and localization. We developed methods to obtain transcriptome-scale structural information and protein binding on individual 3' mRNA isoforms in vivo. Strikingly, near-identical mRNA isoforms can possess dramatically different structures throughout the 3' UTR. Analyses of identical mRNAs in different species or refolded in vitro indicate that structural differences in vivo are often due to trans-acting factors. The level of Pab1 binding to poly(A)-containing isoforms is surprisingly variable, and differences in Pab1 binding correlate with the extent of structural variation for closely spaced isoforms. A pattern encompassing single-strandedness near the 3' terminus, double-strandedness of the poly(A) tail, and low Pab1 binding is associated with mRNA stability. Thus, individual 3' mRNA isoforms can be remarkably different physical entities in vivo. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, indicating biological function.
| S-EPMC6289678 | biostudies-literature
Project description:Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. In this study, we employed a comprehensive multiomics strategy encompassing Ribosome profiling, spike-in normalized RNA-seq, SMPAT-seq, and CAGE-seq to gain mechanistic insight into Pab1's role in Saccharomyces cerevisiae. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2Δ), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2Δ, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2∆, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo at normal mRNA levels. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.
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Project description:Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. In this study, we employed a comprehensive multiomics strategy encompassing Ribosome profiling, spike-in normalized RNA-seq, SMPAT-seq, and CAGE-seq to gain mechanistic insights into Pab1's role in Saccharomyces cerevisiae. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2∆), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2∆, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2∆, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo at normal mRNA levels. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.
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Project description:Cytoplasmic poly(A)-binding protein (PABPC, yeast Pab1) is involved in various stages of mRNA post-transcriptional control, including translation initiation, translation termination, and mRNA decay regulation. Due to the multi-functional properties of PABPC, attempts to define a specific role for PABPC in vivo by deleting, depleting, overexpressing, or mutating the protein has led to conflicting models of PABPC's function. To specifically assess direct and indirect consequences of deleting PABPC, we generated and analyzed RNA-Seq, ribosome profiling, and mass spectrometry data from yeast cells lacking Pab1.
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Project description:Cytoplasmic poly(A)-binding protein (PABPC, yeast Pab1) is involved in various stages of mRNA post-transcriptional control, including translation initiation, translation termination, and mRNA decay regulation. Due to the multi-functional properties of PABPC, attempts to define a specific role for PABPC in vivo by deleting, depleting, overexpressing, or mutating the protein has led to conflicting models of PABPC's function. To specifically assess direct and indirect consequences of deleting PABPC, we generated and analyzed RNA-Seq, ribosome profiling, and mass spectrometry data from yeast cells lacking Pab1.
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Project description:Cytoplasmic poly(A)-binding protein (PABPC, yeast Pab1) is involved in various stages of mRNA post-transcriptional control, including translation initiation, translation termination, and mRNA decay regulation. Due to the multi-functional properties of PABPC, attempts to define a specific role for PABPC in vivo by deleting, depleting, overexpressing, or mutating the protein has led to conflicting models of PABPC's function. To specifically assess direct and indirect consequences of deleting PABPC, we generated and analyzed RNA-Seq, ribosome profiling, and mass spectrometry data from yeast cells lacking Pab1.
2023-06-09 | PXD041495 | Pride
Project description:Nonsense mediated mRNA decay (NMD) is a translation-dependent surveillance pathway that eliminates RNAs with short open reading frames (ORFs) and long 3' untranslated regions (UTRs). Upf1 is the key protein of this degradation pathway and we were interested to purify the associated RNAs. Moreover we have done purifications of two proteins from the general mRNA turnover in yeast : Pab1 and Lsm1 that both bind RNAs poly(A) tails. We have also done a purification with a protein from the small ribosomal subunit (40S) in order to have a look at the translated RNAs.
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2024-12-02 | PXD047579 | Pride