Regulation of alternative splicing by a novel BRR2 interactor C9ORF78

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). Overall design: Identification of C9orf78 RNA targets with FLASH, using GFP as a negative control

RNA解旋酶BRR2（SNRNP200）是剪接体的关键重塑因子之一。本研究证实其与C9ORF78存在直接相互作用——后者是一种功能尚不明确的蛋白质，预测其整体为高度内在无序蛋白。我们解析了冷冻电镜（cryo-EM）结构，揭示C9ORF78与剪接体B复合物蛋白FBP21如何环绕BRR2的C端解旋酶结构域结合，且二者的结合具有互斥性。通过蛋白质组学与RNA紫外交联实验，我们证实C9ORF78可结合于剪接体。经小干扰RNA（siRNA）介导的C9ORF78敲低实验显示，特定靶标前体mRNA（pre-mRNA）的可变剪接发生改变，其中部分改变依赖于其与BRR2的相互作用。尤为重要的是，C9ORF78调控了大量可变3'剪接位点，这一调控过程可能通过其与人类PRP22（DHX8）的额外相互作用得以实现。整体实验设计：以绿色荧光蛋白（GFP）作为阴性对照，采用FLASH技术鉴定C9orf78的RNA靶标。