The transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis and cytoplasmic decay
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ABSTRACT: To function as a system, transcription and post-transcriptional stages must communicate - the underlying mechanism of which is still little studied. Here we focus on S. cerevisiae genes, transcription of which is regulated by Sfp1 that binds their promoters. We show that Sfp1 also binds downstream chromatin regions, thereby changing Pol II configuration, resulting in enhanced Pol II backtracking and possibly Rpb4 dissociation. Unexpectedly, this transcription factor binds >264 transcripts of these genes - near their 3’ ends – and regulates their deadenylation and stability. The interaction of Sfp1 with these mRNAs is controlled by the promoter and it occurs concomitantly with its dissociation from the chromatin. Collectively, we propose that Sfp1 accompanies Pol II and binds the RNA co-transcriptionally. It then accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1 co-transcriptional binding imprints the mRNA fate and serves as a paradigm for posttranscriptional regulation of specific mRNAs by the transcription apparatus.
Project description:Rpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., Pol II, eIF3, Pat1) both temporarily and spatially, Rpb4/7 regulates the major stages of the mRNA lifecycle. Here we show that Rpb4/7 can undergo over 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTMs repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. A mutagenesis approach in residues that undergo PTMs suggests that temporal Rpb4 PTMs regulate its interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis despite its normal association with Pol II, whereas the other affects both mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay (‘mRNA buffering’) and the cell’s capacity to respond to the environment. Taken together, we propose that temporal Rpb4/7 PTMs are involved in cross talks among the various stages of the mRNA lifecycle.
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.
Project description:RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to shuttle from the nucleus to the cytoplasm, and to function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, and evidence arguing against an important cytoplasmic role. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis (cDTA) to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus Rpb4 is a bona fide Pol II core subunit which functions mainly in mRNA synthesis.
Project description:Understanding how transcriptional programs help to coordinate cell growth and division is an important unresolved problem. Here we report that the nutrient- and stress-regulated transcription factor Sfp1 is rate-limiting for expression of a large suite of genes involved in yeast cell growth, including ribosomal protein, ribosome biogenesis, and snoRNA genes. Remarkably, the spectrum of Sfp1 transcription effects is concordant with a combination of chromatin immunoprecipitation and chromatin endogenous cleavage binding analyses, which together provide evidence for two distinct modes of Sfp1 promoter binding, one requiring a co-factor and the other a specific DNA-recognition motif. In addition to growth-related genes, Sfp1 binds to and regulates the promoters of key G1/S regulon genes and that of MRS6, whose product regulates Sfp1 nuclear localization. Our findings suggest that Sfp1 acts as a master regulator of cell growth and cell size by coordinating the expression of genes implicated in mass accumulation and cell division.
Project description:We set out to determine how Sfp1 binding targets evolved over time. We sampled our species of interest to include one pre-whole genome duplicaton (WGD) species containing SFP1A (K. lactis), one post-WGD species containing both SFP1 and SFP1PL (N. castellii), two post-WGD species containing only SFP1 (S. cerevisiae and S. paradoxus) and performed ChIP for each Sfp1 homolog in each species using an antibody that recognizes a conserved subdomain of Sfp1 common to both Sfp1, Sfp1a, and Sfp1pl. Because Sfp1pl was also present in N. castellii, a second antibody was used that is more specific to Sfp1pl (denoted as IP3). We also performed ChIP in SFP1 deletion mutants in each species tested, as a control."
Project description:Yeast RNA polymerase (Pol) II consists of a ten-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immuno-precipitation coupled to high-resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle. Keywords: ChIP-chip
Project description:In Saccharomyces cerevisiae growth, size control and cell cycle progression are strictly coordinated and regulated according to the nutritional conditions. In particular, ribosome biogenesis appears a key event in this regulatory network. SFP1 encodes a zinc-finger protein promoting the transcription of a large cluster of genes involved in ribosome biogenesis. It has been suggested that Sfp1 is a cell size modulator acting at Start. To better study the regulatory role of Sfp1 and its putative involvement in cell size and cycle control, we analysed the behaviour of an sfp1 null mutant strain and of an isogenic reference strain growing in chemostat cultures. This approach allowed us to analyze both strains at the same specific growth rate, thus eliminating the secondary effects due to the slow growing phenotype that the sfp1 null mutant shows in shake flask. We studied glucose(anaerobic)- and ethanol(aerobic)-limited cultures, as paradigms of two different metabolic states. Major alterations of the transcriptional profile were observed during growth on glucose, while no significant differences were observed when comparing ethanol growing cultures. In particular, in the former growth condition, Sfp1 appears involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Keywords: global transcriptional profile, genetic modification, ribosome biogenesis
Project description:Yeast RNA polymerase (Pol) II consists of a ten-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immuno-precipitation coupled to high-resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle. Keywords: ChIP-chip Comparison of Rpb3 vs. Rpb4/7 genome-wide occupancy profiles. Data obtained from independent ChIP-chip experiments on two yeast strains: S288C Rpb3TAP vs. Rpb7-TAP strain and W303 Rpb3-TAP vs. wild type strain (IP with the monoclonal antibody for Rpb4/7). Biological replicates W303 strains: 1 x Rpb3-TAP, 1 x wt with Rpb4/7 antibody, independently grown and harvested. Biological replicates S288C strains: 3 x Rpb3-TAP, 2 x Rpb7-TAP, independently grown and harvested. One replicate per array.
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. Here we investigate the effect of ∆rrp4 on the potential chromatin association of the RNA binding protein Puf3 to chromatin in S. cerevisiae.