Alternative splicing control over naïve and primed pluripotency

Abstract

A fundamental step in embryo development is the transition through pluripotency. There are two known sequential states of pluripotency termed naïve and primed pluripotency. Considerable efforts have been made to characterize these two cell identities using gene expression, protein, metabolic and epigenetic profiling; here we explore an additional regulatory layer, at the level of alternative pre-mRNA splicing (AS). We performed deep coverage RNA sequencing on embryo-derived stem cell lines and identified a regulated AS landscape between the two states involving over 900 splicing events. These events show distinct cis-regulatory features and can also be observed in vivo and in the equivalent human pluripotency states. Differential gene expression analyses helped to identify two regulatory axes that contribute to shape these AS landscapes: the Rbmx/RbmxL2 and the Rbm47/Esrp axes. We further performed a high-throughput screen to test the effect of 1,500 single knockdowns of RNA binding proteins and chromatin modifiers on naïve-to-primed AS switches. This led us to the identification of Qki as an additional master naïve-to-primed AS regulator. Functional assays show that depletion of these splicing regulators affect the differentiation dynamics of embryonic stem cells. Overall, our results provide new insights into the regulatory programs governing naïve and primed pluripotency and the gain of differentiation potential.