RRC ID 64246
Author Kilchert C, Kecman T, Priest E, Hester S, Aydin E, Kus K, Rossbach O, Castello A, Mohammed S, Vasiljeva L.
Title System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA-protein complexes.
Journal Genome Res
Abstract Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA-protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA-protein crosslinking data provide information on the organization of RNA-protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3' end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well.
Volume 30(7)
Pages 1012-1026
Published 2020-7-1
DOI 10.1101/gr.257006.119
PII gr.257006.119
PMID 32554781
Resource
Yeast FY9118