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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of the effect of PUF3 deletion in mRNA translation in yeast

ABSTRACT: The Puf family of RNA-binding proteins regulates gene expression by controlling protein synthesis or stimulating RNA decay. Puf3p is one of six related proteins in Saccharomyces cerevisiae characterised as targeting and regulating decay of nuclear-encoded mRNAs specifying mitochondrial proteins. Much prior work has examined how Puf3p regulates COX17 mRNA. We undertook a genome-wide approach to quantitatively assess the impact of loss of PUF3 on gene expression using polysome profiling. Our translatomic study suggests that loss of Puf3p has widespread, but modest, impact on mRNA translation.

INSTRUMENT(S): AB 5500xl Genetic Analyzer

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: David Talavera

PROVIDER: E-MTAB-3406 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Analysis of the effect of the deletion of PUF3 gene in yeast

Project description:The Puf family of RNA-binding proteins regulate gene expression by controlling protein synthesis or stimulating RNA decay. Puf3p is one of six related proteins in Saccharomyces cerevisiae characterised as targeting and regulating decay of nuclear-encoded mRNAs specifying mitochondrial proteins. Much prior work has examined how Puf3p regulates COX17 mRNA. We undertook a genome-wide approach to quantitatively assess the impact of loss of PUF3 on gene expression. Comparing transcript levels in wild-type and puf3∆ cells revealed that that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only some target mRNAs.

2016-03-31 | E-MTAB-3407 | biostudies-arrayexpress

Proteomics analysis of the effect of deletion of PUF3 on yeast via LC-MSMS.

Project description:The Puf family of RNA-binding proteins regulate the expression of genes by controlling protein synthesis or stimulating RNA decay. Puf3p is one of six Puf-family members in Saccharomyces cerevisiae. Puf3p has been characterised previously as regulating mRNA decay of nuclear mRNAs that encode for mitochondrial proteins. We undertook a series of genome wide approaches to identify an expanded set of Puf3p target mRNAs and to quantitatively assess the impact of loss of PUF3 on the expression control in actively growing wild type and puf3Δ strains. Here we report our quantifications of relative protein levels. The data suggest that loss of Puf3p only impacts on a small proportion of the proteins encoded by its mRNA targets.

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Analysis of the role of yeast Puf3p in translation control

Project description:The Puf family of RNA-binding proteins regulate gene expression by controlling protein synthesis or stimulating RNA decay. Puf3p is one of six related proteins in Saccharomyces cerevisiae characterised as targeting and regulating decay of nuclear-encoded mRNAs specifying mitochondrial proteins. Much prior work has examined how Puf3p regulates COX17 mRNA. We undertook genome-wide approaches to define an expanded set of Puf3p target mRNAs. In agreement with prior studies, our sequencing of affinity-purified Puf3p-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. We also found an additional 720 mRNA targets that predominantly encode proteins that enter the nucleus.

2016-03-31 | E-MTAB-3413 | biostudies-arrayexpress

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Project description:Puf3 is a RNA-binding protein, a member of the conserved Puf-protein family. Combining different functional genomics data, we have analyzed the role of Puf3 in post-transcriptional gene regulation in S. pombe. We present data on Puf3 interacting proteins and regulatory mRNA targets.

2022-03-23 | PXD006318 | Pride

Rpb4 and Puf3 imprint and post-transcriptionally control stability of a common set of mRNAs in yeast

Project description:Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, a crosstalk where mRNA decay machinery impacts transcription and transcription machinery influences mRNA stability. Rpb4, and likely the dimer Rpb4/7 seem to be the central components of the RNA pol II governing these processes. In this work we unravel more precisely the molecular mechanisms participated by Rpb4 that mediates the posttranscriptional events regulating mRNA imprinting and stability. We analysed genome-wide, by RIP-seq, the association of Rpb4 with mRNAs and demonstrated that it targets a broad population of about 1400 transcripts. A group of mRNAs are also targets of the RPB, Puf3. We demonstrated that Rpb4 and Puf3, physically, genetically and functionally interact and cooperate to imprint and regulate stability of this group of mRNA. We also demonstrated, by the first time, that Puf3 associates with the chromatin, in an Rpb4-dependent manner. Our data also point to Rpb4 as the key element of the RNA pol II that interplay mRNA synthesis, imprinting and stability, in cooperation with RBPs.

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Project description:Affinity purification of S. cerevisiae or N. crassa Puf3 from S. cerevisiae cells and identification of associated RNAs by microarray

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Redefining the translational status of 80S monosomes

Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.Â We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.

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PUF3 RNAi in bloodstream-form trypanosomes

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