RNA binding proteins (RBPs) dynamically interact with specific target RNAs, acting as a primary response to extracellular signalling cues. They are key determinants of gene expression at the post-transcriptional level. Despite this, the role of RBPs in the regulation of TGF-β signalling largely remains to be elucidated. In cardiac endothelial cells, TGF-β induces a more mesenchymal-like state (endothelial to mesenchymal transition, EndoMT). In the heart, this is crucial for normal development, however, it is also implicated in a growing number of cardiac pathologies in postnatal life.                  

Using RNA interactome capture, we found that TGF-β drives widespread changes in the binding of a substantial proportion of the endothelial RNA interactome. Interestingly, changes in the RNA binding activity of a number of key RBPs correlated with the onset of TGF-β driven EndoMT. To highlight their importance in regulating TGF-β signalling at the post-transcriptional level, we focussed on two TGF-β regulated RBPs with opposing changes in RNA binding activity upon stimulation with TGF-β.

The binding of the RBP hnRNP H1 was significantly increased following TGF-β stimulation. Interestingly, we found that hnRNP H1 is protective for endothelial cell function, with loss of hnRNP H1 exacerbating EndoMT development (i.e. increasing mesenchymal gene-expression, but reducing angiogenic function). RNA immunoprecipitation in conjunction with RNA sequencing showed that hnRNP H1 distinctly increases its affinity for mRNAs encoding proteins with functions involved in TGF-β signalling and EndoMT. We revealed, for instance, that hnRNP H1 counteracts EndoMT by binding to and increasing SMAD6 abundance, a classical inhibitor of TGF-β signalling, while at the same time binding and decreasing the expression of Col1a1, a core pro-mesenchymal gene. Increased binding of hnRNPH1 to the mRNAs encoding Smad6 and Col1a1 was also verified in situ in myocardial endothelial cells of mice during cardiac pressure overload versus sham operation.

Conversely, the binding of Csde1 was significantly downregulated by TGF-β. We found that Csde1 is also protective for endothelial cell function, with Csde1 knock-down resulting in an EndoMT-like phenotype despite the absence of TGF-β. TGF-β stimulation resulted in less interaction between Csde1 and RNAs encoding mesenchymal genes, resulting in their increased expression. We specifically revealed that Csde1 prevents the expression of mesenchymal genes such as Col5a1 and Itga3, and that the TGF-β driven decrease in binding to these targets results in their increased abundance. Decreased binding of Csde1 to the mRNAs encoding Itga3 and Col5a1 was also verified in situ in myocardial endothelial cells of mice during cardiac pressure overload versus sham operation.           

Together, we have shown that RBPs are key determinants of the endothelial response to TGF-β signalling at the post-transcriptional level. Both hnRNP H1 and Csde1 are important in maintaining endothelial cell function, with the TGF-β driven increase in RNA binding by hnRNP H1 offsetting EndoMT development in a negative feed-back mechanism, while the TGF-β driven decrease in RNA binding by Csde1 facilitating the development of EndoMT. Specifically increasing the RNA binding affinity of either protein in the endothelium may therefore provide unique opportunities to therapeutically prevent the development of EndoMT and pathological fibrosis.