Regulation of alternative splicing by C9ORF78, a novel BRR2 interactor II

The RNA helicase BRR2 (SNRNP200) is one of the key remodeling factors of the spliceosome. Here we show its direct interaction with C9ORF78, a poorly characterized protein predicted to be largely intrinsically disordered. We present cryo-EM structures showing how C9ORF78 and the spliceosomal B-complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 and that binding of the two proteins is mutually exclusive. C9ORF78 associates with the spliceosome, as we confirm via proteomics and RNA UV-crosslinking. An siRNA mediated C9ORF78 knockdown reveals changes in alternative splicing of specific target pre-mRNAs, which in part depend on its interaction with BRR2. In particular, C9ORF78 regulates a substantial number of alternative 3’ splice sites, which might be facilitated through an additional interaction with human PRP22 (DHX8). HEK293T cells were transfected either with empty vector, C9ORF78WT or C9ORF78mut and 2h lather either with a CTRL siRNA ( empty vector C) or an siRNA targeting C9orf78 (empty vector S, C9ORF78WT W or C9ORF78mut M); 72h after transfection RNA was extracted and analyzed by RNA-Seq.