Surveying the global landscape of post-transcriptional regulators
TL;DR: The yeast RNA-binding protein Mrn1p is especially important in linking cell wall biogenesis with mitochondrial homeostasis, and it regulates these two cellular compartments in a manner that is responsive to carbon source and cell stress.
Abstract: Author(s): Reynaud, Kendra Keilani | Advisor(s): Ingolia, Nicholas T | Abstract: At all stages of a messenger RNA’s lifecycle, it is covered in RNA-binding proteins. These proteins regulate an RNA transcript’s splicing and processing in the nucleus, its export from the nucleus into the cytoplasm, its localization and translation in the cytoplasm, and its eventual turnover and decay. Despite knowing the identities of roughly 700 RNA-binding proteins in budding yeast, the role in RNA regulation that many of these proteins perform remains unclear. Here we present two studies that are aimed at the functional characterization of proteins that regulate post-transcriptional gene expression. In the first study, we devised a high-throughput tethering assay for the characterization of proteins on a proteome-wide scale. This novel assay provides domain-level resolution for the functional regions of proteins and identifies their regulatory activity in a quantitative manner. In the second study, we characterized the yeast RNA-binding protein Mrn1p and found that it is a dynamic regulator of post-transcriptional regulation that functions through mRNA turnover. Mrn1p is especially important in linking cell wall biogenesis with mitochondrial homeostasis, and it regulates these two cellular compartments in a manner that is responsive to carbon source and cell stress. Together, we present two studies that provide new functional information about yeast RNA binding proteins, with broad implications for a better understanding of post-transcriptional gene expression.
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01 Jun 2018
TL;DR: It is shown that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5′ untranslated regions, and this program has a role in meiosis, in which a marked decrease in the levels of Ded 1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded2p is repressed.
Abstract: The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.
70 citations
30 Jun 2021
TL;DR: In this article, the authors show that Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo, and they further demonstrate that the effect of puf1-induced post-transcriptional repression does not obligatorily require dissociation of YpK1-phosphorylated Puf 1 from a transcript.
Abstract: Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3'-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.
3 citations
References
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TL;DR: The popular MEME motif discovery algorithm is now complemented by the GLAM2 algorithm which allows discovery of motifs containing gaps, and all of the motif-based tools are now implemented as web services via Opal.
Abstract: The MEME Suite web server provides a unified portal for online discovery and analysis of sequence motifs representing features such as DNA binding sites and protein interaction domains. The popular MEME motif discovery algorithm is now complemented by the GLAM2 algorithm which allows discovery of motifs containing gaps. Three sequence scanning algorithms—MAST, FIMO and GLAM2SCAN—allow scanning numerous DNA and protein sequence databases for motifs discovered by MEME and GLAM2. Transcription factor motifs (including those discovered using MEME) can be compared with motifs in many popular motif databases using the motif database scanning algorithm Tomtom. Transcription factor motifs can be further analyzed for putative function by association with Gene Ontology (GO) terms using the motif-GO term association tool GOMO. MEME output now contains sequence LOGOS for each discovered motif, as well as buttons to allow motifs to be conveniently submitted to the sequence and motif database scanning algorithms (MAST, FIMO and Tomtom), or to GOMO, for further analysis. GLAM2 output similarly contains buttons for further analysis using GLAM2SCAN and for rerunning GLAM2 with different parameters. All of the motif-based tools are now implemented as web services via Opal. Source code, binaries and a web server are freely available for noncommercial use at http://meme.nbcr.net.
7,733 citations
TL;DR: The construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.
Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.
4,310 citations
TL;DR: A significant comparison to the structural classification database that led to the creation of 825 new families based on their set of uncharacterized families (EUFs) was carried out and Pfam entries were connected to the Sequence Ontology (SO) through mapping of the Pfam type definitions to SO terms.
Abstract: The last few years have witnessed significant changes in Pfam (https://pfam.xfam.org). The number of families has grown substantially to a total of 17,929 in release 32.0. New additions have been coupled with efforts to improve existing families, including refinement of domain boundaries, their classification into Pfam clans, as well as their functional annotation. We recently began to collaborate with the RepeatsDB resource to improve the definition of tandem repeat families within Pfam. We carried out a significant comparison to the structural classification database, namely the Evolutionary Classification of Protein Domains (ECOD) that led to the creation of 825 new families based on their set of uncharacterized families (EUFs). Furthermore, we also connected Pfam entries to the Sequence Ontology (SO) through mapping of the Pfam type definitions to SO terms. Since Pfam has many community contributors, we recently enabled the linking between authorship of all Pfam entries with the corresponding authors' ORCID identifiers. This effectively permits authors to claim credit for their Pfam curation and link them to their ORCID record.
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