Opposing roles of miR‐294 and MBNL1/2 in shaping the gene regulatory network of embryonic stem cells

Abstract Alternative pre‐mRNA splicing plays important roles in regulating self‐renewal and differentiation of embryonic stem cells (ESCs). However, how specific alternative splicing programs are established in ESCs remains elusive. Here, we show that a subset of alternative splicing events in ESCs is dependent on miR‐294 expression. Remarkably, roughly 60% of these splicing events are affected by the depletion of Muscleblind‐Like Splicing Regulator 1 and 2 (Mbnl1/2). Distinct from canonical miRNA function, miR‐294 represses Mbnl1/2 through both posttranscriptional and epigenetic mechanisms. Furthermore, we uncover non‐canonical functions of MBNL proteins that bind and promote the expression of miR‐294 targets, including Cdkn1a and Tgfbr2, thereby opposing the role of miR‐294 in regulating cell proliferation, apoptosis, and epithelial–mesenchymal transition (EMT). Our study reveals extensive interactions between miRNAs and splicing factors, highlighting their roles in regulating cell type‐specific alternative splicing and defining gene expression programs during development and cellular differentiation.