Department of Molecular Biosciences, University of Texas, Austin, TX, USA.
Sci Adv. 2024 Sep 6;10(36):eadq0350. doi: 10.1126/sciadv.adq0350.
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.
RNA 聚合酶 II 依赖其大亚基中的重复序列结构域 (YSPTSPS) 来协调转录。虽然羧基末端七肽重复序列丝氨酸-2/丝氨酸-5 的磷酸化已得到充分证实，但丝氨酸-4 的作用仍然是个谜。矛盾的是，尽管在暂停、延伸、终止和信使 RNA 加工中具有功能意义，但丝氨酸-4 的磷酸化仅在转录末端位点检测到。我们的研究表明，尽管丝氨酸-5 的磷酸化在转录开始时会阻碍丝氨酸-4 的磷酸化检测，但这种磷酸化可以被选择性去除。随后的蛋白质组学分析鉴定了许多通过丝氨酸-4 磷酸化招募到转录中的蛋白质，这些蛋白质以前归因于丝氨酸-2。丝氨酸-4 磷酸化的丧失大大降低了丝氨酸-2 磷酸化，揭示了这两个标记之间的交叉对话。最后，丝氨酸-4 磷酸化的功能分析强调了它在增殖基因的替代 3'-末端加工中的作用。我们的发现揭示了这个进化上保守的磷酸化标记的真实基因组位置，并促使人们重新评估羧基末端结构域的功能分配。
