A Screen for RNA-Binding Proteins in Yeast Indicates Dual Functions for Many Enzymes

Abstract

Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation.

The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

“…While Rtr1p does not have homology with any known RNA binding domain and an x-ray crystal structure of Rtr1p also did not necessarily reveal an RNA binding domain, it may be that the binding of Rtr1p to its target sequence, if direct, may occur in a noncanonical fashion and possibly through a disordered region . Recent evidence suggests that a large population of previously unrecognized RBPs exist among metabolic enzymes and other factors not specifically recognized as being involved in RNA metabolism (Scherrer et al 2010;Tsvetanova et al 2010). Utilizing two approaches to UV crosslinking of RBPs to RNA, over 300 new RBPs have been discovered in HeLa cells, many of which are involved in metabolic processes (Castello et al 2012).…”

A versatile assay for RNA-binding proteins in living cells

“…The dual fluorescence RNA-binding assay described here can be used to validate the in vivo RNA-binding activity of candidate RBPs identified by system-wide approaches (Scherrer et al 2010;Tsvetanova et al 2010;Baltz et al 2012;Castello et al 2012;Klass et al 2013;Kwon et al 2013;Mitchell et al 2013). We assessed the RNA-binding activity of three previously known RBPs (MOV10, hnRNPC, and cytoplasmic poly(A)-binding protein [PABP]), three recently discovered RBPs (FAM98A, FAM32A, and enolase 1 [ENO1]), and three negative controls (H2B, β-actin [ACTB], eGFP).…”

“…The complexity of protein-RNA networks and their regulation substantially increased by the recent discovery of hundreds of previously unidentified noncanonical RBPs using high-content approaches (Scherrer et al 2010;Tsvetanova et al 2010;Baltz et al 2012;Castello et al 2012;Klass et al 2013;Kwon et al 2013;Mitchell et al 2013).…”

RNA regulons and the RNA-protein interaction network

“…In the worm Caenorhabditis elegans and in the fruitfl y Drosophila melanogaster approximately 2 % and in humans about 5 % of the protein coding genes are annotated as ' RNA binding ' (UniProt/ Swissprot release 2012_2). However, it is likely that the number of RBPs is even higher in light of the recent discoveries of many potentially novel RBPs (15,16) . Furthermore, several of the RNA-binding domains, such as the RRM, underwent drastic amplifi cation during animal evolution (12) , in parallel with the evolution of highly specifi c post-transcriptional processes, such as alternative splicing, allowing an increased genetic diversity from a limited repertoire of genes.…”