Gene Center Munich and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany.
Gene Center Munich and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany; Department of Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Mol Cell. 2015 Jun 18;58(6):1079-89. doi: 10.1016/j.molcel.2015.04.004. Epub 2015 May 7.
Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5'-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5' end. Thus, the RNA 5' end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5'-exonucleases during early transcription.
加帽是前体 mRNA 加工的第一步，产生的 5'-RNA 帽对于 mRNA 的剪接、输出、翻译和稳定性都是必需的。加帽功能上与 RNA 聚合酶 (Pol) II 的转录偶联，但偶联机制尚不清楚。我们发现，成熟的 5'-三磷酸末端的未加工 RNA 对于有效结合帽酶 (CE) 至关重要。转录 Pol IIp-CE 复合物催化加帽的前两个步骤，通过质谱分析、冷冻电镜和蛋白质交联分析，揭示了转录偶联的前体 mRNA 加帽的分子基础。CE 与 Pol II 壁结合，并跨越 RNA 出口隧道的末端，将 CE 的活性位点定位，以便顺序结合从 Pol II 中出来的 RNA 5' 末端。因此，当 RNA 从 Pol II 中出来时，其 5' 末端会触发自身的加帽，以确保在早期转录过程中，RNA 能免受 5'-核酸外切酶的侵害。
