RNAs associated with Upf1, Pab1, Rpl16a, Lsm1 proteins in yeast
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ABSTRACT:
PROVIDER: PRJNA673832 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12965700.fastq.gz | Fastqsanger.gz | |||
| SRR12965701.fastq.gz | Fastqsanger.gz | |||
| SRR12965702.fastq.gz | Fastqsanger.gz | |||
| SRR12965703.fastq.gz | Fastqsanger.gz | |||
| SRR12965704.fastq.gz | Fastqsanger.gz |
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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.
2022-10-01 | GSE160642 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts.
2025-06-13 | GSE284490 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts.
2025-06-13 | GSE283053 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved RNA degradation pathway that is involved in development, resistance to viral infections and evolution. Upf1, a core NMD factor, is associated with a large variety of cellular RNA in complex ribonucleoprotein particles. We characterized NMD complexes previously by affinity purification of tagged protein followed by mass spectrometry. Here, we extend our observations to proteins that are present in NMD complexes, but for whom the interaction with NMD factors is mediated by RNA. We purified tagged versions of Lsm1, Lsm7, Pat1, Dhh1 and Pab1 and assessed the presence and amount of NMD and RNA-related proteins in each purification. As expected from our previously published results, Upf1 was present in these purifications, suggesting novel hypotheses about the role of Pab1 and the Lsm1-7 complexes in RNA degradation.
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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 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:We performed mass-spectrometry analysis of proteins co-purifying with the Pab1, the B55 regulatory subunit of the PP2A protein phosphatase complex. Pab1 was tagged with YFP at the C-terminus and expressed from its endogenous promoter at its chromosomal locus. After one hour of nitrogen starvation, Pab1-YFP was pulled down and co-purifying proteins were analysed by mass-spectrometry. Our results revealed the presence of all components of PP2A protein complexes including Paa1, the catalytic subunits Ppa1, Ppa2, and Ppa3.
2024-12-02 | PXD047697 | Pride
Project description:Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In S. cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the mutually exclusive organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex whereby its CH, RecA and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.
2025-10-15 | GSE307461 | GEO
Project description:Comparison of WT, xrn1 delta and upf1 delta strains were used in a tiling array to yield genomic regions regulated by these proteins The supplementary CHP files record either the signal in log2 space or the p-values in linear space, per TAS output. The CHP files are further divided between UPF1 delta vs. WT and XRN1 delta vs. WT.
2008-08-21 | GSE11621 | GEO
Project description:The nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic cytoplasmic surveillance pathway that degrades aberrant mRNAs carrying premature termination codons (PTC). In yeast, the long distance between the stop codon and the poly(A) tail dictates mRNA detection by NMD factors Upf1, Upf2 and Upf3, and trigger mRNA decay, independent of deadenylation. While Upf1 binds a wide range of mRNAs beyond PTC-containing substrates, the mechanism of its recruitment on non-PTC-containing mRNAs is unknown. Using Nanopore sequencing of Upf1-bound mRNAs, we discovered that known PTC-containing NMD targets have long poly(A) tails, confirming that Upf1 binding occurs rapidly prior to deadenylation. Strikingly, the largest category specifically bound to Upf1 consisted of mRNAs without PTC harboring short poly(A) tails, corresponding to well-translated and stable transcripts. We showed that the degradation of this subpopulation depends on the three Upf factors, revealing a hitherto unknown function of Upf1 and NMD cofactors in mRNA decay.
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Project description:Like-SM proteins (Lsm) assemble into heptameric complexes that are conserved in eukaryotes. The Lsm2-8 complex is involved in the function of spliceosomal RNAs, whereas the Lsm1-7/Pat1 complex associates with decapping complexes in the cytoplasm. It was proposed that the role of Lsm1-7 is to recruit the decapping machinery to unstable mRNA that are rapidly deadenylated. However, the role of deadenylation in RNA stability has been challenged by recent studies in yeast and other organisms. To better understand the potential role of Lsm1 in the degradation of unstable transcripts, we investigated the effects of rapid depletion of Lsm1 in an inducible degron system on transcripts degraded by decapping activation during nonsense-mediated mRNA decay (NMD). In contrast to previous studies that used deletion of LSM1, Lsm1 depletion, led to a slight increase in the abundance of NMD substrates, similar to what is observed following a minimal perturbation of decapping.
2025-07-31 | GSE303057 | GEO