MBNL1-mediated Regulation of Differentiation RNAs Promotes Myofibroblast Transformation and the Fibrotic Response

The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodeling. To better understand this process we performed a genome-wide screen in fibroblasts, which identified the RNA-binding protein muscleblind-like 1 (MBNL1) as a potent regulator of myofibroblast differentiation. MBNL1 promoted transformation of fibroblasts into myofibroblasts, while loss of Mbnl1 abrogated myofibroblast differentiation and impaired the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Conditional fibroblast-specific MBNL1 transgenic mice showed a fibrotic phenotype in the absence of injury. Mechanistically, MBNL1 directly bound to and regulated the alternative splicing and stability of a network of myofibroblast differentiation specific genes, such as the nodal transcriptional regulator serum response factor (SRF). CRISPR-Cas9 mediated gene editing of the MBNL1 binding site in Srf impaired myofibroblast differentiation. These data establish a new RNA-dependent paradigm through MBNL1 that underlies global myofibroblast differentiation, the fibrotic response, and tissue wound healing. To elucidate the potential mechanism whereby MBNL1 programs myofibroblast differentiation, we analyzed the expression of polyadenylated transcripts by RNA sequencing (RNAseq) from Mbnl1+/+ and Mbnl1-/- MEFs that were infected with AdMBNL1, Adβgal (negative control), or treated with TGFβ (positive control for myofibroblast differentiation). To more rigorously identify RNAs that are directly regulated by MBNL1, RNA immunoprecipitation (RIP) assays were performed on rat cardiac fibroblasts infected with Adβgal (control) or a Flag epitope tagged AdMBNL1 construct (AdMBNL1-Flag). Flag antibody resin was used to immunoprecipitate all RNAs that bound to MBNL1, followed by RNAseq analysis.