Abstract
RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.
Highlights
RNA-binding proteins (RBPs) are implicated in diverse human disorders, including X-linked intellectual disability (XLID), which accounts for $16% of all cases of intellectual disability (Ellison et al, 2013)
To characterize this genomic lesion, we obtained an iPSC line derived from a healthy human male and truncated the C terminus of RBMX using CRISPR-Cas9 to replicate what is observed in patients with Shashi-XLID
Using gene set enrichment analysis (GSEA) with hallmark gene sets, we identified a few pathways to be dysregulated in DRGG1 cells, including the epithelial-mesenchymal transition (EMT) pathway, the KRAS signaling pathway, estrogen response, hypoxia, and the p53 pathway (Figure S1B)
Summary
RNA-binding proteins (RBPs) are implicated in diverse human disorders, including X-linked intellectual disability (XLID), which accounts for $16% of all cases of intellectual disability (Ellison et al, 2013). More than 75% of RBPs are arginine methylated, which affects RBP function, including alternative splicing regulation (Thandapani et al, 2013). Alternative splicing is highly involved in brain development, modulating each step of lineage commitment, neurogenesis, and neuron function (Su et al, 2018). RNA sequencing (RNA-seq) comparing developing human brain-derived Neural progenitor cell (NPC) and neuron samples revealed that alternative splicing governs cell fate and splicing factors are dynamically regulated during development (Zhang et al, 2016). RBMX was proposed as a candidate gene for the XLID subtype termed the Shashi-XLID syndrome for a family pedigree with seven affected males (Shashi et al, 2015). RBMX knockdown in rat primary hippocampal neurons causes a decrease in dendritic spine density, indicating its important role in the regulation of synaptic activity (Zhang et al, 2012)
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