TY - JOUR

T1 - Quantitative proteomic analysis reveals concurrent RNA-protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae

AU - Klass, Daniel M

AU - Scheibe, Marion

AU - Butter, Falk

AU - Hogan, Gregory J

AU - Mann, Matthias

AU - Brown, Patrick O

PY - 2013/6

Y1 - 2013/6

N2 - 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 Puf3. 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 Puf3 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.

AB - 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 Puf3. 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 Puf3 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.

KW - Cluster Analysis

KW - Models, Biological

KW - Nucleocytoplasmic Transport Proteins

KW - Protein Binding

KW - Proteomics

KW - RNA Processing, Post-Transcriptional

KW - RNA, Messenger

KW - RNA-Binding Proteins

KW - Reproducibility of Results

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Trans-Activators

U2 - 10.1101/gr.153031.112

DO - 10.1101/gr.153031.112

M3 - Journal article

C2 - 23636942

VL - 23

SP - 1028

EP - 1038

JO - Genome Research

JF - Genome Research

SN - 1088-9051

IS - 6

ER -