RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on serine-2/serine-5 of the carboxyl-terminal heptad repeats is well established, threonine-4’s role remains enigmatic. Paradoxically, threonine-4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and messenger RNA processing. Our investigation revealed that threonine-4 phosphorylation detection was obstructed by flanking serine-5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via threonine-4 phosphorylation, which previously were attributed to serine-2. Loss of threonine-4 phosphorylation greatly reduces serine-2 phosphorylation, revealing a cross-talk between the two marks. Last, the function analysis of the threonine-4 phosphorylation highlighted its role in alternative 3′-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the carboxyl-terminal domain.

中文翻译：

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RNA Pol II 上的 Thr 4 磷酸化发生在调节 3′-末端加工的早期转录中


RNA 聚合酶 II 依赖于其最大亚基内的重复序列结构域 （YSPTSPS） 来编排转录。虽然羧基末端七肽重复序列的丝氨酸-2/丝氨酸-5 上的磷酸化已得到充分证实，但苏氨酸-4 的作用仍然是个谜。矛盾的是，苏氨酸-4 磷酸化仅在转录末端位点后检测到，尽管它在功能上与暂停、延伸、终止和信使 RNA 加工有关。我们的研究显示，苏氨酸-4 磷酸化检测在转录开始时受到侧翼丝氨酸-5 磷酸化的阻碍，可以选择性地去除。随后的蛋白质组学分析确定了许多通过苏氨酸-4 磷酸化募集到转录中的蛋白质，这些蛋白质以前归因于丝氨酸-2。苏氨酸-4 磷酸化缺失会大大降低丝氨酸-2 磷酸化，从而揭示两个标记之间的串扰。最后，苏氨酸-4 磷酸化的功能分析突出了其在促增殖基因内替代 3′ 末端加工中的作用。我们的研究结果揭示了这种进化上保守的磷酸化标记的真实基因组位置，并促使重新评估羧基末端结构域的功能分配。