Abstract
The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF65-SF1-RNA complex which occurs at the 3′ end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions.
Highlights
Gene expression is subject to a host of regulatory mechanisms, expanding the way in which cells can regulate their protein composition and setting the bases for the complexity of morphology and connectivity of neuronal cells
Using an intronic acceptor RNA substrate, we showed that phosphorylation of SF1 favors the formation of a ternary RNA-U2AF65-SF1 complex
We analyzed the expression of KIS and of the splicing factors SF1, U2AF65 and SAP155 during brain development
Summary
Gene expression is subject to a host of regulatory mechanisms, expanding the way in which cells can regulate their protein composition and setting the bases for the complexity of morphology and connectivity of neuronal cells. In addition to control at the level of gene transcription, the steps of pre-mRNA splicing, trafficking and translation in the gene expression process are exquisitely regulated [1]. The splicing factor SF1 is phosphorylated in vivo on two serines within a highly conserved SPSP motif and substrates for the protein kinase KIS in vitro [3]. We previously observed that the dual phosphorylation of SF1 on its SPSP motif enhances SF1 interaction with U2AF65 [3]. Using an intronic acceptor RNA substrate, we showed that phosphorylation of SF1 favors the formation of a ternary RNA-U2AF65-SF1 complex. This suggested a function of SF1 phosphorylation by KIS in the recognition of the 39 splice site. Additional functions have been proposed for KIS, including the phosphorylation of p27kip, stathmin and PAM [11,12,13,14]
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