Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
Mol Cell. 2021 Mar 4;81(5):998-1012.e7. doi: 10.1016/j.molcel.2020.12.018. Epub 2021 Jan 12.
Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.
在许多生物中，前体 mRNA 加工步骤与转录紧密协调。为了确定哺乳动物细胞中转录延伸和 3' 端形成如何与共转录剪接整合，我们在小鼠红细胞生成过程中进行了单个新生 RNA 的长读测序和精确运行测序 (PRO-seq)。剪接没有伴随着转录暂停，并且在 RNA 聚合酶 II (Pol II) 位于 3' 剪接位点 (3'SS) 的 75-300 个核苷酸内时被检测到，通常在下游外显子的转录过程中。有趣的是，数百个内含子显示出丰富的剪接中间体，表明两个催化步骤之间可能发生剪接延迟。总体而言，同一转录本内的内含子之间的剪接效率相关，内含子保留与 3' 端切割效率低下有关。值得注意的是，一种由地中海贫血患者衍生的突变引入了一个隐蔽的 3'SS，提高了单个β-珠蛋白转录本的剪接和 3' 端切割效率，证明了这两个共转录过程之间的功能偶联是产生有功能的基因产物的决定因素。
