Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them. Overall design: Transcription elongation rates were determined within gene bodies and dwonstream of termination sites by mapping pol II positions after release of a DRB block or after inhibiting initiation with triptolide. Elongation rates were estimated under conditions where termination was inhibited by a dominant negative mutants of the exonuclease Xrn2 or the PP! nuclear targeting subunit, PNUTS. In addition Spt5 phosphorylation was measured by ChIP and found to correlate closely with chages in elongation rate.

转录速度的调控会影响诸多共转录过程，而该调控机制目前仍未得到充分解析。我们的研究表明，PNUTS-PP1磷酸酶（PNUTS-PP1 phosphatase）是RNA聚合酶II（RNA pol II）延伸速率的负调控因子。破坏PP1结合能力的PNUTS W401A突变，会导致全基因组范围内的转录加速，该现象与Spt5延伸因子（Spt5 elongation factor）的过度磷酸化相关。在多聚腺苷酸化位点（poly(A) site）的紧邻下游区域，RNA聚合酶II的转录速率会从>2千碱基对/分钟降至<1千碱基对/分钟，这一变化与Spt5的去磷酸化过程密切相关。RNA聚合酶II的减速以及Spt5的去磷酸化，需要依赖对多聚腺苷酸化位点的识别以及PNUTS-PP1复合物（PNUTS-PP1 complex），而该复合物同时也是转录终止所必需的。上述研究结果提出了一种全新的转录终止模型——“坐鸭鱼雷”（sitting duck torpedo）机制：该机制中，由PNUTS-PP1介导的多聚腺苷酸化位点依赖性转录减速，以及Spt5的去磷酸化过程，是将RNA聚合酶II转化为Xrn2终止核酸外切酶（Xrn2 terminator exonuclease）可识别的有效靶标所必需的。因此，Spt5与其细菌同源物NusG具有功能相似性，二者均可调控RNA聚合酶与追踪其行进的终止因子之间的动力学竞争。实验整体设计：通过在解除DRB阻滞或用雷公藤内酯（triptolide）抑制转录起始后，定位RNA聚合酶II的基因组位置，我们测定了基因本体内部以及终止位点下游区域的转录延伸速率。在利用核酸外切酶Xrn2或PP1核靶向亚基PNUTS的显性负突变体抑制转录终止的条件下，我们同样估算了转录延伸速率。此外，我们通过染色质免疫沉淀（ChIP）检测了Spt5的磷酸化水平，发现其与转录延伸速率的变化呈现高度相关性。