MBNL proteins repress ES-cell-specific alternative splicing and reprogramming

Abstract

Previous investigations of the core gene regulatory circuitry that controls embryonic stem cell (ESC) pluripotency have largely focused on the roles of transcription, chromatin and non-coding RNA regulators1–3. Alternative splicing (AS) represents a widely acting mode of gene regulation4–8, yet its role in regulating ESC pluripotency and differentiation is poorly understood. Here, we identify the Muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon AS events that are differentially regulated between ESCs and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ESC-like AS pattern for approximately half of these events, whereas over-expression of MBNL proteins in ESCs promotes differentiated cell-like AS patterns. Among the MBNL-regulated events is an ESC-specific AS switch in the forkhead family transcription factor FOXP1 that controls pluripotency9. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells (iPSCs) during somatic cell reprogramming.

The Calcineurin Variant CnAβ1 Controls Mouse Embryonic Stem Cell Differentiation by Directing mTORC2 Membrane Localization and Activation

“…We also provide insight into the regulation of CnA post-transcriptional processing that leads to expression of CnAβ1. We show for the first time that CnA splicing is controlled, at least in part, by Mbnl1, which has a similar role to that of CnAβ1 in the differentiation of ESCs (Han et al, 2013). Mbnl1 was previously shown to favor ESC differentiation by controlling AS of different genes.…”

“…2SA and B). It has been recently described that muscleblind like (Mbnl) proteins are major splicing regulators in ESCs that repress pluripotency and promote differentiation (Han et al, 2013). To investigate the potential regulation of CnA splicing by Mbnl1, we first explored available CLIP-Seq datasets for this trans-regulatory factor obtained from myoblasts and differentiated tissues (Wang et al, 2012).…”

“…To investigate the potential regulation of CnA splicing by Mbnl1, we first explored available CLIP-Seq datasets for this trans-regulatory factor obtained from myoblasts and differentiated tissues (Wang et al, 2012). These datasets have been previously used to identify Mbnl1 targets shared between C2C12 and mESCs (Han et al, 2013). We identified two Mbnl1 binding sites in CnA's intron 12-13 in C2C12 myoblasts that were absent in adult differentiated tissues (Fig.…”

“…The presence of Mbnl1 binding marks in C2C12 cells correlates with CnAβ1 expression, which is higher in myoblasts than in fully differentiated tissues . To determine the role of Mbnl proteins in the regulation of CnAβ1 in mESCs we downregulated both Mbnl1 and Mbnl2 using previously reported siRNAs (Han et al, 2013) (Fig. 1G).…”

Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning

“…RBPs play a crucial role in regulating splicing, having an impact on a wide variety of developmental stages such as stem cell differentiation 46 and tissue development 47 . We predicted binding scores at junctions near exons that are putatively regulated by known splicing regulators that exhibit large changes in splicing when knocked down, including: Nova (neuro-oncological ventral antigen 1 and 2), PTBP1 (polypyrimidine tract binding protein 1), Mbnl1/2 (muscleblind-like protein), hnRNP C (heterogeneous nuclear ribonucleoprotein C) and cytotoxic TIA (T-cell intracellular antigen-like 1).…”