RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-β to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells

Abstract

Background: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. Methods: Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. Results: Characterization of TGF-β–regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. Conclusions: Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

CSDE1: a versatile regulator of gene expression in cancer

“…iCLIP peak counts from references ( 47 ) and ( 49 ) were averaged across replicates and scaled for comparison. ( C ) Venn diagram comparing CSDE1 RNA targets in mouse cardiac endothelial cells (MCEC) ( 74 ), melanoma ( 47 ) and either proliferating or senescent PMK ( 49 ). A core of 229 targets is highlighted.…”

“…In order to obtain a global view of CSDE1 targets, we compared the transcripts bound to endogenous CSDE1 in three large-scale studies performed in melanoma cells, primary mouse keratinocytes (PMK, either under proliferating or senescent conditions), and mouse cardiac endothelial cells (MCEC) ( 47 , 49 , 74 ). Although thousands of transcripts were bound by CSDE1 in each condition, a common set of 229 targets was found, supporting the prevalent context-specific nature of CSDE1 interactions with RNA (Figure 3C ).…”