RNA binding proteins (RBPs) are central to the regulation of gene expression at the post-transcriptional level. Through dynamic interactions with their target RNAs, RBPs determine changes in the translation, stability, splicing and localisation of transcripts and serve as a key response mechanism to changes in cellular conditions. Methodological advances such as RNA interactome have enabled the global identification of the RNA bound proteome under physiological and pathophysiological conditions, providing invaluable insights into the roles of RBPs in health and disease.

Using RNA interactome capture, we identified the RNA bound proteome of cardiac endothelial cells and determined changes in RNA binding activity upon TGF-β stimulation. TGF-β is the key driver of endothelial-mesenchymal transition (EndMT), whereby endothelial cells undergo a loss of endothelial phenotype and change towards more mesenchymal-like cells, which has been implicated in a range of cardiac pathologies in post-natal life. While the mechanisms through which TGF-β regulates this process are well detailed at the transcriptional level (E.g. SMAD activation), the post-transcriptional mechanisms (i.e. changes mediated through RBPs) largely remained to be elucidated.

We identified several key RBPs with TGF-β regulated RNA binding activity correlating with progression of EndMT. TGF-β stimulation resulted in a significant increase in the RNA binding activity of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), a predominantly nuclear RBP with diverse functions in the regulation of RNA metabolism, as well as distinct changes in its localisation. Furthermore, by knock-down or overexpression of hnRNP H1, we found that it plays a protective role in the maintenance of endothelial phenotype and offsets development of EndMT.

Using both RNA immunoprecipitation and RNA sequencing, we identified the target RNAs to which hnRNP H1 dynamically binds and regulates upon TGF-β stimulation. We found sequence specific TGF-β driven changes in binding to distinct subsets of RNAs with functions related to the regulation of endothelial function and establishment of a mesenchymal phenotype and confirmed that hnRNP H1 knock-down results in an upregulation the abundance of EndMT related RNAs.

We visualised the interaction between hnRNP H1 and Col1a1 and Smad6 RNA at the molecular level both in vitro and in in vivo mouse heart sections following pathological overload. This revealed a significant increase in the nuclear interactions between hnRNP H1 and Col1a1 and Smad6 dynamically upon TGF-β stimulation in vitro, and a specific enrichment for these interactions in endothelial cells compared to other cardiac cell types in vivo upon induction of pathological heart remodelling. Interestingly, we found hnRNP H1 expression is enriched in endothelial cells compared to other cardiac cell types (cardiomyocytes and fibroblasts) and hnRNP H1 expression is upregulated in endothelial cells following pathological overload.

Together, we have identified that the RNA binding protein hnRNP H1 exhibits TGF-β driven changes in its RNA binding activity in cardiac endothelial cells. We found that hnRNP H1 plays a protective role in maintaining the endothelial-like phenotype and preventing development of EndMT. Furthermore, we have shown that hnRNP H1 dynamically binds to Col1a1 and Smad6 RNA and these interactions are enriched in cardiac endothelial cells in vivo upon pathological overload.