Defining the RNA interactome by total RNA-associated protein purification

Molecular Systems Biology
Vadim ShchepachevDavid Tollervey

Abstract

The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP...Continue Reading

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Methods Mentioned

BETA
immunoprecipitation
PCR
electrophoresis
GTPase
GTPases
affinity purification
iCLIP
acetylation

Software Mentioned

pyGTF2bedGraph
search
Novoindex
David
imputeLCMD R package
Proteome discoverer
MS excel
Decoy database builder
Novoalign
RNP XL

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