RNA-seq analysis of the budding yeast ctk1? and rad6? strains and prp45(1-169) ctk1? and prp45(1-169) rad6? double mutant strains

Prp45 is a budding yeast NineTeen-Complex associated factor, which plays a role in pre-mRNA splicing. Because its human ortholog, SNW1/SKIP, is known to functionally and physically interact with factors involved in transcription elongation and chromatin modifications, it was suggested to be one of the coupling factors which functionally connect splicing and transcription. To determine whether the yeast Prp45 has this function as well we tested genetic interactions between prp45(1-169) allele and deletions of histone modifiers and transcription elongation regulators. RNA-seq analysis was performed to address the changes in transcription and/or splicing in strains deleted in ctk1 and rad6, a kinase phosphorylating serine 2 of the C-terminal domain of RNA Polymerase II and an E2 enzyme for H2BK123 monoubiquitination, respectively, and the corresponding double mutants with prp45(1-169). Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.

Prp45是出芽酵母中与十九复合物（NineTeen-Complex）相关的因子，其功能参与前mRNA剪接（pre-mRNA splicing）过程。鉴于其人类同源物SNW1/SKIP已被证实可在功能与物理层面上，与转录延伸（transcription elongation）及染色质修饰（chromatin modifications）相关因子发生相互作用，因此Prp45被认为是连接剪接与转录的功能性耦合因子之一。为明确酵母Prp45是否同样具备该功能，我们检测了prp45(1-169)等位基因（allele）与组蛋白修饰因子（histone modifiers）、转录延伸调控因子（transcription elongation regulators）缺失突变体之间的遗传相互作用（genetic interactions）。我们通过RNA测序（RNA-seq）分析，探究了ctk1缺失菌株、rad6缺失菌株，以及二者与prp45(1-169)构建的对应双突变体中的转录与/或剪接变化情况。其中，ctk1为可磷酸化RNA聚合酶II C端结构域（C-terminal domain）丝氨酸2位点的激酶，rad6则是负责H2BK123单泛素化（monoubiquitination）的E2酶（E2 enzyme）。总RNA提取采用苯酚-氯仿（phenol-chloroform）提取法结合MasterPure酵母RNA纯化试剂盒（Epicentre）进行；核糖体RNA去除（ribodepletion）、文库制备及测序工作均由BGI Genomics完成。