Regulation of alternative splicing by C9ORF78, a novel BRR2 interactor II. 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). Overall design: 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.

RNA解旋酶（RNA helicase）BRR2（SNRNP200）是剪接体（spliceosome）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：C9ORF78是一种功能尚未得到充分表征的蛋白质，被预测为整体上高度内在无序蛋白（intrinsically disordered protein）。我们解析了冷冻电镜（cryo-EM）结构，揭示了C9ORF78与剪接体B复合物蛋白FBP21如何环绕结合于BRR2的C端解旋酶结构域，且二者与BRR2的结合相互排斥。通过蛋白质组学和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可调控大量可变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：将HEK293T细胞分别转染空载体、野生型C9ORF78（C9ORF78WT）或突变型C9ORF78（C9ORF78mut）；转染2小时后，再分别转染对照（CTRL）siRNA（对应空载体对照组C）或靶向C9orf78的siRNA（分别对应空载体沉默组S、野生型C9ORF78沉默组W及突变型C9ORF78沉默组M）；转染72小时后提取RNA，通过RNA测序（RNA-Seq）进行分析。