The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease

Abstract

SummaryRNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs) in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

“…Recently, mRNA-protein interactome capture to profile the set of proteins bound to polyadenylated (poly[A]) RNAs across different organisms has become popular [7][8][9][10][11][12][13][14]. These studies confirmed hundreds of previously known RBPs, but likewise identified many novel RBPs, such as metabolic enzymes and others, whose function in post-transcriptional gene regulation is still enigmatic [11,12,14,15].…”

Capturing the interactome of newly transcribed RNA

“…Short-labeling RICK has revealed a potential link between metabolic enzymes/factors and nascent RNAs. Many metabolic enzymes/factors had been identified as bona fide RBPs in oligo(dT) capture studies 1,39 , but it was not known that some of them may interact with nascent RNAs. Short-labeling RICK could also be useful to differentiate RBPs interacting with newly transcribed RNAs from those interacting with steady-state RNAs.…”

Intersections of post-transcriptional gene regulatory mechanisms with intermediary metabolism

“…However, studies over the past three decades have revealed that these housekeeping enzymes may also have non-canonical regulatory roles. For instance, certain enzymes have the capacity to bind specific mRNAs, and regulate their expression post-transcriptionally [133][134]. One of the first moonlighting enzymes to be identified, thymidylate synthase (TS), normally serves a critical role in de novo nucleotide biosynthesis by catalyzing the conversion of dUMP to dTMP.…”