Functional Diversification of SRSF Protein Kinase to Control Ubiquitin-Dependent Neurodevelopmental Signaling

Abstract

Summary Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.

A RNF12-USP26 amplification loop promotes germ cell specification and is disrupted in urogenital disorders

“…TOKAS patients suffer other congenital anomalies, including defects in urogenital development/hypogenitalism. In order to investigate molecular mechanisms that may underpin syndromic features of TOKAS, we analysed our previously reported RNA-SEQ dataset (Bustos et al, 2020) to determine developmental gene expression signatures that are positively regulated by RNF12 in male mouse embryonic stem cells (mESCs) cultured in Leukemia Inhibitory Factor (LIF) and Fetal Bovine Serum (FBS; Figure 1A). Amongst RNAs that are induced by WT RNF12 (Figure 1A), Gene Ontology (GO) term analysis identifies significant enrichment of RNAs relating to reproduction, sexual development and gametogenesis (Figure 1B).…”

“…A) RNA-sequencing (RNA-SEQ) data comparing RNF12-deficient mouse embryonic stem cells (mESCs; Rlim −/y ) transfected with either empty vector (Control) or those expressing wild-type (WT) RNF12 was reported previously (Bustos et al, 2020). RNAs were ranked according to fold-change increase in expression upon RNF12 reconstitution (left).…”

“…A cohort of the most dynamically regulated RNAs implicated in gametogenesis, including Usp26, Usp9y, Tdrd12, Dazl, Dppa3 , was identified and is summarised (Table 1). RNA expression was analysed in control RNF12 knock-in mESCs (WT-KI) and RNF12 knock-in mESCs harbouring a catalytic mutant that impairs E3 ubiquitin ligase activity (W576Y-KI) (Bustos et al, 2020) leading to accumulation of ubiquitylated RNF12 substrates including the REX1/ZFP42 transcriptional repressor (Figure 1D). Gametogenesis RNAs identified in our RNA-SEQ dataset are expressed in mESCs cultured in LIF/FCS, but this is significantly reduced in RNF12 W576Y-KI mESCs with the exception of Rhox5 (Figure 1E).…”

“…Next, we set out to identify the mechanism by which RNF12 drives gametogenesis gene expression. The REX1 transcriptional repressor has been identified as a key RNF12 substrate in developmental systems (Bach, 2012; Bustos et al, 2020; Gontan et al, 2012; Gontan et al, 2018). Ubiquitylation and degradation of REX1 relieves transcriptional repression to promote expression of the long non-coding RNA Xist , which in turn initiates imprinted X-chromosome inactivation (Gontan et al, 2012; Gontan et al, 2018).…”

“…Recent work by our lab has uncovered an RNF12-dependent signalling pathway that controls expression of neurodevelopmental genes (Bustos et al, 2020), suggesting a potential molecular mechanism by which functional disruption of RNF12 by variants found in TOKAS patients could result in intellectual disability. However, deregulation of neurodevelopmental genes is unlikely to explain other syndromic features characteristic of TOKAS.…”