Quantitative proteomic analysis reveals concurrent RNA–protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae

Abstract

A growing body of evidence supports the existence of an extensive network of RNA-binding proteins (RBPs) whose combinatorial binding affects the post-transcriptional fate of every mRNA in the cell-yet we still do not have a complete understanding of which proteins bind to mRNA, which of these bind concurrently, and when and where in the cell they bind. We describe here a method to identify the proteins that bind to RNA concurrently with an RBP of interest, using quantitative mass spectrometry combined with RNase treatment of affinity-purified RNA-protein complexes. We applied this method to the known RBPs Pab1, Nab2, and Puf 3. Our method significantly enriched for known RBPs and is a clear improvement upon previous approaches in yeast. Our data reveal that some reported protein-protein interactions may instead reflect simultaneous binding to shared RNA targets. We also discovered more than 100 candidate RBPs, and we independently confirmed that 77% (23/30) bind directly to RNA. The previously recognized functions of the confirmed novel RBPs were remarkably diverse, and we mapped the RNA-binding region of one of these proteins, the transcriptional coactivator Mbf1, to a region distinct from its DNA-binding domain. Our results also provided new insights into the roles of Nab2 and Puf 3 in post-transcriptional regulation by identifying other RBPs that bind simultaneously to the same mRNAs. While existing methods can identify sets of RBPs that interact with common RNA targets, our approach can determine which of those interactions are concurrent-a crucial distinction for understanding post-transcriptional regulation.

Evolutionarily Conserved Polyadenosine RNA Binding Protein Nab2 Cooperates with Splicing Machinery To Regulate the Fate of Pre-mRNA

“…Notably, ZC3H14 colocalizes with nuclear speckles that contain the splicing factor SC-35 (48,52,53), suggesting a potential role for ZC3H14 in processing events such as splicing. In support of a link between Nab2/ZC3H14 and splicing, a recent proteomic study found that Nab2 associates with several splicing factors in an RNA-dependent manner (54).…”

Computational Tools for Investigating RNA-Protein Interaction Partners

“…With such large numbers of RPBs, each of which binds multiple mRNA and/or ncRNA targets, another difficult task will be to identify which combinations of RBPs determine specific post-transcriptional fates of individual mRNAs and ncRNAs. Progress in this direction was demonstrated in a quantitative proteomic analysis in S. cerevisiae, which identified sets of RBPs that bind simultaneously to common RNA targets [50]. Computational tools for constructing and interrogating RNA-protein interaction networks and for integrating RPIs into existing gene and protein interaction networks will be needed.…”

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).…”