Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892, USA.
Cell. 2021 May 27;184(11):2878-2895.e20. doi: 10.1016/j.cell.2021.04.012. Epub 2021 May 11.
The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.
RNA 聚合酶和剪接体的活性负责人类细胞中 mRNA 丰度和同工型组成的异质性。然而，这些兆道尔顿酶复合物在协同作用于内源性基因时的动态尚未被描述。在这里，我们建立了一个准基因组规模的平台，用于实时观察单个新生 RNA 分子的合成和加工动力学。我们发现所有观察到的基因都显示转录爆发。我们还观察到单个细胞中单条内含子的内含子去除的动力学变化很大，这与确定性剪接位点选择不一致。U2AF 复合物的转录组范围足迹分析、新生 RNA 分析、长读测序和套索测序进一步揭示了内含子中广泛存在的随机递归剪接。我们提出并验证了一个统一的理论模型，以解释转录和普遍存在的随机剪接位点选择的一般特征。
