Widespread Cotranslational Formation of Protein Complexes

Caia D S Duncan,Juan Mata,Dieter A Wolf

doi:10.1371/journal.pgen.1002398

Abstract

Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly). We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA–binding domains, we found that ∼38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication. Both proteins interact with Cdc2p and are key cell cycle regulators. We obtained analogous results with proteins with different structures and cellular functions (kinesins, protein kinases, transcription factors, proteasome components, etc.). We showed that copurification of a bait protein and of specific mRNAs was dependent on the presence of the proteins encoded by the interacting mRNAs and on polysomal integrity. These results indicate that these observed associations reflect the cotranslational interaction between the bait and the nascent proteins encoded by the interacting mRNAs. Therefore, we show that the cotranslational formation of protein–protein interactions is a widespread phenomenon.

Highlights

The majority of cellular proteins function as subunits in larger protein complexes
A protein is purified together with associated RNAs, and the mRNAs are identified using DNA microarrays. When this method is applied to proteins associated with polysomes, it allows the identification of mRNAs cotranslationally associated with the bait protein. Using this technique we recently showed that the Rng3p myosin-specific chaperone associates cotranslationally with all five myosin heavy chains in the fission yeast Schizosaccharomyces pombe [6]
To investigate if cells use cotranslational assembly to form complexes, we identified mRNAs associated with specific proteins

Summary

Introduction

The majority of cellular proteins function as subunits in larger protein complexes. very little is known about how protein complexes form in vivo. A number of recent studies have shown that the use of immunoprecipitation coupled with microarray analysis (RIp-chip, for Ribonucleoprotein Immunoprecipitation analysed with DNA chips) can be used to study cotranslational pathways involved in protein biosynthesis [6,7,8]. In this approach, a protein is purified together with associated RNAs, and the mRNAs are identified using DNA microarrays. The protein-RNA interactions were dependent on active translation, suggesting that the complex between these proteins was formed cotranslationally [7] Apart from these few examples, very little is known about the prevalence of cotranslational assembly in the formation of protein complexes. Systematic approaches to identify and characterise this phenomenon (such as RIp-chip) have not been applied to large numbers of proteins

Methods

Results

Discussion

Conclusion