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Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.

ABSTRACT: The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs--most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.

SUBMITTER: Tsvetanova NG

PROVIDER: S-EPMC2937035 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.

Tsvetanova Nikoleta G NG Klass Daniel M DM Salzman Julia J Brown Patrick O PO

PloS one 20100910 9

The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs--most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-pr ...[more]

PMID: 20844764

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Project description:UNLABELLED:In the model eukaryote Saccharomyces cerevisiae, the transcription factor Cst6p has been reported to play important roles in several biological processes. However, the genome-wide targets of Cst6p and its physiological functions remain unknown. Here, we mapped the genome-wide binding sites of Cst6p at high resolution. Cst6p binds to the promoter regions of 59 genes with various biological functions when cells are grown on ethanol but hardly binds to the promoter at any gene when cells are grown on glucose. The retarded growth of the CST6 deletion mutant on ethanol is attributed to the markedly decreased expression of NCE103, encoding a carbonic anhydrase, which is a direct target of Cst6p. The target genes of Cst6p have a large overlap with those of stress-responsive transcription factors, such as Sko1p and Skn7p. In addition, a CST6 deletion mutant growing on ethanol shows hypersensitivity to oxidative stress and ethanol stress, assigning Cst6p as a new member of the stress-responsive transcriptional regulatory network. These results show that mapping of genome-wide binding sites can provide new insights into the function of transcription factors and highlight the highly connected and condition-dependent nature of the transcriptional regulatory network in S. cerevisiae IMPORTANCE:Transcription factors regulate the activity of various biological processes through binding to specific DNA sequences. Therefore, the determination of binding positions is important for the understanding of the regulatory effects of transcription factors. In the model eukaryote Saccharomyces cerevisiae, the transcription factor Cst6p has been reported to regulate several biological processes, while its genome-wide targets remain unknown. Here, we mapped the genome-wide binding sites of Cst6p at high resolution. We show that the binding of Cst6p to its target promoters is condition dependent and explain the mechanism for the retarded growth of the CST6 deletion mutant on ethanol. Furthermore, we demonstrate that Cst6p is a new member of a stress-responsive transcriptional regulatory network. These results provide deeper understanding of the function of the dynamic transcriptional regulatory network in S. cerevisiae.

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Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.

Publications

Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.

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