A Highly Conserved GAD-1 Is Required for Pre-mRNA Splicing and Transcription Elongation by Forming Spliceosome with NineTeen Complex

Pre-mRNA splicing requires assembly of spliceosome that consists of hundreds of factors forming various dynamic complexes with or without small nuclear RNAs (snRNAs). Systematic identification of the splicing factors remains a significant challenge especially in vivo. In our genetic screening for the factors required for division asynchrony during Caenorhabditis elegans embryogenesis, we identified a highly conserved but uncharacterized essential protein, GAD-1, that is necessary for setting cell cycle length of intestine progenitors, a function that is shared with many other factors involved in transcription, pre-mRNA splicing or polyadenylation, suggesting its potential role in mRNA biogenesis. Co-immunoprecipitation followed by mass spectrometry reveals that GAD-1 mainly interacts with non-snRNP type of splicing complex called NineTeen Complex (NTC). Consistent with this, RNA-seq analysis demonstrates pervasive defects in pre-mRNA splicing in gad-1 mutants. Transgenic reporter assay shows its ubiquitous and nuclear expression across developmental stages. Immunostaining of the C-terminal domain of RNA polymerase II demonstrates a GAD-1's role in transcription elongation. In agreement with this, depletion of GAD-1 and its interacting partners inhibits expression of both ubiquitous and tissue-specific genes, supporting that both GAD-1 and many of its interacting proteins are novel components of NTC or its associated spliceosome. Taken together, we identify GAD-1 and its multiple interacting partners as novel components of spliceosome in vivo through which they regulate pre-mRNA splicing and transcription elongation.  Overall design: Mix-stage embryo samples were collected from strain BW1943 (gad-1(ct226) dpy-11(e224) V) at 16 °C and 25 °C by bleaching. Total RNAs were extracted using TRIzol (Invitrogen) following the manufacturer's instructions. mRNA purification and fragmentation, cDNA synthesis, end repairing, adapters ligation, and DNA fragment enrichment were performed using Illumina's TruSeq Stranded mRNA Library Preparation Kit based on the Kit's manual. Each library was sequenced to obtain pair-end (2×150 bps) reads using Illumina HiSeq 2500. We obtained approximately 20 million reads of high-quality score (>30 mean quality score) on average per library.

前mRNA（pre-mRNA）剪接需要剪接体（spliceosome）的组装，剪接体由数百种因子组成，可结合或不结合小核RNA（small nuclear RNAs，snRNAs）形成多种动态复合物。剪接因子的系统性鉴定仍是一项重大挑战，尤其是在体内（in vivo）环境中。在针对秀丽隐杆线虫（Caenorhabditis elegans）胚胎发生过程中分裂异步性所需因子的遗传筛选中，我们鉴定出一种高度保守但尚未被研究的必需蛋白质GAD-1，其对于维持肠道祖细胞的细胞周期长度是必需的；该功能与许多参与转录、前mRNA剪接或多聚腺苷酸化的其他因子一致，提示其在mRNA生物发生中可能发挥作用。免疫共沉淀（Co-immunoprecipitation）结合质谱分析结果显示，GAD-1主要与一类名为十九复合物（NineTeen Complex，NTC）的非snRNP型剪接复合物相互作用。与此一致的是，RNA测序（RNA-seq）分析显示，gad-1突变体中存在广泛的前mRNA剪接缺陷。转基因报告基因实验显示，GAD-1在各个发育阶段均呈广泛表达且定位于细胞核内。针对RNA聚合酶II（RNA polymerase II）C端结构域的免疫荧光染色实验证实，GAD-1参与转录延伸过程。与此相符的是，敲低GAD-1及其互作蛋白会抑制组成型表达基因与组织特异性基因的表达，这支持GAD-1及其多数互作蛋白均为NTC或其相关剪接体的新型组分。综上，我们在体内鉴定出GAD-1及其多个互作蛋白为剪接体的新型组分，它们通过该复合物调控前mRNA剪接与转录延伸。

整体实验设计：通过漂白法分别在16℃与25℃条件下，从菌株BW1943（gad-1(ct226) dpy-11(e224) V）中收集混合阶段胚胎样本。按照试剂厂商说明书，使用TRIzol（Invitrogen）提取总RNA。参照Illumina TruSeq链特异性mRNA文库制备试剂盒的操作手册，完成mRNA纯化与片段化、cDNA合成、末端修复、接头连接及DNA片段富集等步骤。使用Illumina HiSeq 2500测序平台对每个文库进行双端（2×150 bp）测序。每个文库平均获得约2000万条高质量测序读段（平均质量得分>30）。