Identification of RNA binding and processing targets of RBMX protein and their role in maintaining genome stability

Abstract

RBMX is part of a family of RNA binding proteins that regulate pre-mRNA splicing. RBMX is conserved in all vertebrates and has a key role in splicing regulation. RBMX has a homologue called RBMY on the Y chromosome that is sometimes deleted in infertile men. RBMX has several extra-copies originating from retrotransposition (like ELAV in Drosophila) retroposed copies on autosomes include RBMXL2 that represses cryptic splice sites during male meiosis. RBMX has been implicated in cancer development and progression. When the presence of RBMX is altered in cells, it affects the activity of genes involved in DNA damage, suggesting that RBMX acts as a tumour suppressor. However, the precise role of RBMX in RNA processing, including its target RNAs, are unknown. To investigate RBMX-RNA binding I carried out an iCLIP analysis that suggests RBMX directly binds highly to exons and introns of protein-coding genes. RBMX binding was highest in genes involved in DNA damage and replication. To explore the role of RBMX in splicing, it was depleted from MDA-MB231 cells followed by RNAseq analysis. This showed that RBMX blocks selection of aberrant splice sites within some ultra-long exons that would interfere with genes needed for normal replication fork activity. Target exons include within the ETAA1 (Ewings Tumour Associated 1) gene, where RBMX directly binds to exon 5 to enable full-length exon inclusion by blocking selection of an aberrant 3′ splice site. Branch point mapping of the ETAA1 cryptic exon revealed a branch point closely adjacent to RBMX binding sites. These data suggest that RBMX may act as a steric block to selection of this cryptic ETAA1 branch point by the spliceosome. Preliminary experiments in MDA-MB231 cells have been used to monitor genome integrity after RBMX depletion. Together our iCLIP RBMX binding data and RNA-seq splicing analysis reveal that RBMX is involved in the repression of long exons. Our data helps explain why RBMX is associated with gene expression networks in cancer, DNA replication and sensitivity to genotoxic drugs.