3′-end sequencing of poly(A)+ RNA in wild-type Saccharomyces cerevisiae and a strain carrying a deletion of the RRP6 nuclear exosome catalytic component. Saccharomyces cerevisiae

The nuclear exosome performs critical functions in non-coding RNA processing, and in diverse surveillance functions including the quality control of mRNP formation, and in the removal of pervasive transcripts. Most non-coding RNAs and pervasive nascent transcripts are targeted by the Nrd1p-Nab3p-Sen1p (NNS) complex to terminate Pol II transcription coupled to nuclear exosome degradation or 3´-end trimming. Prior to nuclear exosome activity, the Trf4p-Air2p-Mtr4p polyadenylation complex adds an oligo-A tail to exosome substrates. Inactivating exosome activity stabilizes and lengthens these A-tails. We utilized high-throughput 3´-end poly(A)+ sequencing to identify at nucleotide resolution the 3´ ends targeted by the nuclear exosome, and determine the sites of NNS-dependent termination genome-wide. Overall design: 3´-end mapping of wild-type and various nuclear exosome mutant strains, either using gene knockouts or the anchor away system to conditionally deplete FRB-tagged proteins from the nucleus

核外切体（nuclear exosome）在非编码RNA加工中发挥关键功能，同时参与多种监视过程，包括mRNA-核蛋白复合物（mRNP，messenger ribonucleoprotein）形成的质量控制，以及清除广泛存在的转录本。绝大多数非编码RNA与广泛存在的新生转录本会被Nrd1p-Nab3p-Sen1p（NNS）复合物靶向，以终止与核外切体降解或3'端修剪偶联的RNA聚合酶II（Pol II）转录。在核外切体发挥活性之前，Trf4p-Air2p-Mtr4p多聚腺苷酸化复合物会向外切体底物添加寡聚A尾。抑制核外切体活性可使此类A尾得以稳定并延长。我们采用高通量3'端多聚A+测序技术，以核苷酸分辨率鉴定了核外切体靶向的3'末端，并在全基因组范围内确定了NNS依赖性终止的位点。整体实验设计：对野生型菌株与各类核外切体突变菌株开展3'端定位分析，突变构建方式包括基因敲除，或借助锚定撤离系统（anchor away system）从细胞核中条件性耗竭带有FRB标签的蛋白。