Multiple direct and indirect roles of the Paf1 Complex in elongation, splicing, and histone post-translational modifications [4tU-seq]

Eukaryotes employ a set of conserved transcription elongation factors to modulate the behavior of RNA polymerase II (RNAPII). Disruptions of one such factor, the Paf1 complex (Paf1C), generate subunit-specific phenotypes, including distinct changes to co-transcriptional histone modifications. How individual Paf1C subunits impact transcription and coupled processes remains ambiguous. By comparing conditional depletion and steady-state deletion of Paf1C subunits, we determine direct and indirect contributions of Paf1C to gene expression in Saccharomyces cerevisiae. Through nascent transcript sequencing, RNAPII profiling, and mechanistic modeling of transcription elongation dynamics, we find evidence for unique roles of Paf1C subunits in regulating RNAPII processivity, elongation rate, mRNA stability, transcript splicing, and repression of antisense transcripts. Through genetic suppression, we attribute increased antisense transcription, but not other defects, of a PAF1 mutant to loss of H3 K36 methylation. This work comprehensively analyzes both the immediate and extended roles of Paf1C subunits in transcription elongation and transcript regulation.真核生物依赖一套保守的转录延伸因子，调控RNA聚合酶II（RNA polymerase II, RNAPII）的活性。其中一类核心因子为Paf1复合物（Paf1 complex, Paf1C），当其功能遭到破坏时，会引发亚基特异性表型，包括共转录组蛋白修饰的显著改变。目前，Paf1C各亚基如何影响转录及其偶联生物学过程仍不明确。本研究通过对比Paf1C亚基的条件性耗竭与稳态缺失实验，明确了酿酒酵母（Saccharomyces cerevisiae）中Paf1C对基因表达的直接与间接调控作用。借助新生转录本测序（nascent transcript sequencing）、RNA聚合酶II谱分析以及转录延伸动力学机制建模，我们发现Paf1C各亚基在调控RNA聚合酶II持续合成能力、延伸速率、mRNA稳定性、转录本剪接以及反义转录本抑制等方面存在独特功能。通过遗传抑制实验，我们将PAF1突变体所出现的反义转录上调缺陷（而非其他异常表型），归因于H3 K36甲基化的丢失。本研究全面解析了Paf1C亚基在转录延伸与转录本调控中的即时与长效作用。