Background

RNA binding proteins (RBPs) are key regulators of gene expression at the post-transcriptional level controlling all aspects of RNA metabolism including localisation, translation efficiency, stability and splicing. RNA-protein interactions are highly dynamic; changes in RNA binding activity serve as a primary response to environmental cues, sculpting the cellular proteome to reflect changes in environmental conditions. Development of methodologies such as RNA interactome capture enable the global identification of mRNA bound proteins under physiological conditions, dramatically improving our understanding of the role of RBPs in regulation of cell function. 

The cardiac endothelium is highly heterogeneous, making a key contribution to cardiac function and the development of numerous cardiovascular diseases. The ability of endothelial cells to adopt a change in phenotype towards more mesenchymal characteristics (endothelial to mesenchymal transition, EndMT) is critical to heart development, although it is also implicated in a number of complications such as cardiac fibrosis, atherosclerosis and valvular disease in postnatal life. A number of signaling pathways play a role in the regulation of endothelial cell function and development of EndMT (such as TGF-β signaling); however, the downstream molecular mechanisms governing changes in endothelial phenotype are poorly understood.

Results

Using RNA interactome capture followed by quantitative proteomics, we identified global changes in RNA binding activity of RBPs in cardiac endothelial cells in response to TGF-β stimulation. This revealed 116 RBPs which changed in RNA binding activity upon TGF-β stimulation. Subsequent validation and characterisation of selected RBPs by RNA interactome capture and Western blot analysis revealed many dynamic TGF-β induced changes in RNA binding activity in a dose and time dependent manner correlating with loss of endothelial characteristics and development of EndMT.

Two of these RBPs, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) and Cold Shock Domain Containing E1 (Csde1), were found to bind to RNA in opposing manners upon TGF-β stimulation. Intriguingly, we found that both of these proteins exhibit a protective role for endothelial characteristics by siRNA knock-down, suppressing TGF-β mediated effects and the development of EndMT.

Using RNA immunoprecipitation, we identified the specific RNAs to which both hnRNP H1 and Csde1 bind. Furthermore, we identified nucleotide specific changes in their affinity for their target RNAs upon TGF-β stimulation. This revealed that they each differentially bind distinct subsets of functionally related target RNAs, suggesting that TGF-β stimulates specific changes in their RNA binding activity.

Conclusions

Our data has revealed that TGF-β dynamically regulates the RNA binding activity of a large number of RBPs in cardiac endothelial cells. We have found that two conventional RBPs, hnRNP H1 and Csde1, exhibit dynamic changes in RNA binding activity upon TGF-β stimulation, driven at least in part by sequence specific changes in their affinity for functionally related RNAs.

Interestingly, we have found that the RNA binding activity of both hnRNP H1 and Csde1 is important for the maintenance of the endothelial phenotype. Our current work focuses on understanding the role of these proteins and their RNA binding activity in both endothelial cell function and dysfunction, and their wider implications in disease.