SLAM-seq estimates of mRNA half-life changes in S. cerevisiae strains depleted for Dcp2 and Ccr4/Pop2
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE247954 | GEO | 2023/11/24
REPOSITORIES: GEO
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Project description:miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. AGO1 is a key protein required for miRNA-mediated gene silencing. In animal cells, mRNA degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1 or NOT1. We show that approximately 45% of AGO1-targets are regulated by both CAF1 and NOT1, indicating deadenylation is a widespread effect of miRNA regulation.<br><br>We employed RNA interference using long double-stranded RNAs to deplete cultured S2 cells of AGO1 (CG6671, 2 independent samples), CAF1 (CG5684, 2 independent samples), or NOT1 (CG1884, 3 independent samples). Further, we used Affymetrix oligonucleotide microarrays to analyze expression profiles in these samples. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (3 and 2 independent samples, respectively).<br>
2008-12-03 | E-MEXP-1713 | biostudies-arrayexpress
Project description:Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 function to negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the DCP2 mRNA decapping enzyme has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent pathway.
2021-09-09 | GSE159510 | GEO
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression. 5' monophosphorylated ends of poly(A) RNA from wild-type and dcp2D xrn1D strains were identified in duplicates and triplicates, respectively.
2012-06-17 | E-GEOD-33712 | biostudies-arrayexpress
Project description:Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
2020-04-16 | GSE131408 | GEO
Project description:Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
2020-04-16 | GSE144250 | GEO
Project description:The prevailing model postulates that complete cytoplasmic polyadenosine tail (pA-tail) deadenylation is essential for initiating mRNA decapping and subsequent degradation. To investigate this, we conducted direct RNA sequencing of yeast mRNAs derived from steady-state and stress condition chase experiments. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile pA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNA, which appeared consistent within functional transcript groups and associated with to codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40 % of the transcriptome, singled out this transcript group. While RPG mRNA deadenylation and decay accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating decapping onset, while also demonstrating complex relations between these processes.
2024-09-11 | GSE272785 | GEO
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression.
2012-06-17 | GSE33712 | GEO
Project description:RNA degradation pathways underlie RNA processing, the regulation of RNA levels, and the surveillance of aberrant or poorly functional RNAs in cells. Here we provide transcriptome-wide RNA-binding profiles of 30 general RNA degradation factors in the yeast S. cerevisiae. The profiles define the distribution of factors between RNA classes. They are also consistent with the canonical degradation pathway for closed-loop forming mRNAs after deadenylation. Modeling based on mRNA half-lives suggests that most degradation factors bind already to intact mRNAs, whereas decapping factors are recruited only for mRNA degradation, consistent with decapping being a rate-limiting step. Global analysis suggests that the nuclear surveillance machinery, including the complexes Nrd1/Nab3, TRAMP4, and the exosome, targets aberrant nuclear RNAs, pre-processes snoRNAs, and regulates attenuated genes. Finally, mRNA transcript optimality is the most predictive feature for binding of decapping factors and the Xrn1 exonuclease to mRNA, consistent with rapid 5´-degradation of inefficiently translated mRNAs.
2019-05-28 | GSE128312 | GEO
Project description:mRNA degradation critically contributes to tissue development and function as well as transcription. The CCR4-NOT complex serves as a major deadenylase that initiates mRNA degradation. We used microarrays to identify deregulated genes in the white adipose tissues lacking CNOT3, a core subunit of the CCR4-NOT complex.
2017-01-01 | GSE86930 | GEO
Project description:The evolutionarily conserved CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon histone deacetylase (HDAC) inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified ring finger protein 219 (RNF219) as an acetylation-regulated cofactor. We provide evidence that RNF219 is an active RING-type E3 ubiquitin ligase which stably associates with the CCR4-NOT complex via the NOT9 module through a short linear motif located in the C-terminal low-complexity region of RNF219. Using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 acts as an inhibitor of deadenylation. Accordingly, our transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, independently of its E3 ligase activity. Our results establish RNF219 as a negative regulator of CCR4-NOT-mediated mRNA deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.
2021-12-15 | PXD027237 | Pride