RNA-Binding Proteins (RBPs) are omnipresent partners of cellular RNA. For example, eukaryotic mRNAs dynamically associate with RBPs assisting their transcription, maturation, nuclear to cytoplasmic translocation, localization, translation and degradation. The ‘RNA interactome capture’ method uses in-vivo UV crosslinking followed by oligo(dT) purification and mass spectrometry of enriched peptides. It has led to the identification of hundreds of novel RBPs in cardiomyocytes (Liao et al., Cell Rep 2016, 16:1456–1469). This included multiple metabolic enzymes, given further credence to the RNA-Enzyme-Metabolite (REM) hypothesis, which proposes the existence of regulatory links between gene expression and intermediary metabolism mediated by RNA-binding metabolic enzymes. Possible functions include enzymes ‘moonlighting’ in the regulation of mRNA utilisation or (noncoding) RNAs regulating enzyme function (Castello et al., Trends Endocrinol Metab 2015, 26:746–757).
To explore these scenarios, we focus on prominent cardiomyocyte metabolic RBPs such as enolase, citrate synthase, aconitase-2, malate dehydrogenase and isocitrate dehydrogenase. We are in the middle of implementing a plan that involves the use of in vivo UV or formaldehyde crosslinking to stabilise the REM partners, followed by affinity purification via a GFP tag on the enzyme and identification of RNA binders by deep sequencing; precise binding sites in the RNA will be located by limited degradation and/or reverse transcription stalling approaches. We will further use site-specific mutagenesis of both target RNA and enzyme in HL-1 cells and mice hearts at different developmental stages. Given the significance of metabolic pathways in heart, unravelling REM interaction networks can provide new insights into cardiomyocyte development, biology, and pathophysiology.