FMRP-Regulated Alternative Splicing is Multifactorial and Resembles Splicing Control by MBNL1

Abstract

The neurodevelopmental disorder Fragile X Syndrome (FXS) is often modeled in Fmr1 knockout mice, which results in the loss of the RNA binding protein FMRP. In the brain, FMRP stalls ribosomes on specific mRNAs including Setd2, whose encoded protein catalyzes the epigenetic mark H3K36me3. In the absence of FMRP, SETD2 levels are excessive, which alters the H3K36me3 landscape and secondarily, alternative pre-mRNA splicing. Here we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, hnRNPQ, PTPB1, and MBNL1. FMRP binds to Mbnl1 mRNA and regulates its translation. In Fmr1-depleted cells, Mbnl1 RNA itself is mis-spliced, which results in the loss of a nuclear localization signal (NLS)-containing exon that in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This re-distribution of MBNL1 isoforms in Fmr1-deficient cells likely results in splicing changes in other RNAs. Mbnl1 mis-spicing also occurs in human FXS post-mortem brain. These data link FMRP-dependant impaired translation of splicing factors, such as MBNL1, to altered self-splicing and subcellular localization of the Mbnl1 RNA and protein. Altered expression of splicing factors in the absence of FMRP may cascade into the global dys-regulation of tissue specific splicing observed in Fmr1 deficient cells.