Elucidate the functions of the BET bromodomain protein BRD4 in co-transcriptional splicing

RNA聚合酶II （Pol II）的转录是真核细胞基本细胞过程的基础，并受到严格调控。一个主要的控制点发生在早期延伸之前的Pol II释放到生产延伸。先前的研究已经将BET溴结构域蛋白家族及其成员BRD2、BRD3和BRD4与人类细胞的转录伸长联系起来。我们的研究最近提供的证据表明，BET溴结构域蛋白BRD4是一般5 ' -延伸检查点的基础，有助于组装功能性Pol II延伸复合物并刺激生产延伸。转录延伸与RNA加工紧密耦合并在时间上协调，使RNA在合成时成熟。我们最近发现BRD4是转录延伸和RNA加工之间的分子联系。我们发现BRD4招募3 ' -RNA加工因子在5 ‘ -延伸检查站转录参与Pol II，使基因3 ’端RNA切割（Arnold et al., Mol Cell, 2021）。我们还发现BRD4与剪接体的亚基相互作用，并且急性BET蛋白消融影响了大量基因的剪接模式。这些发现表明BRD4与共转录剪接有关，类似于转录偶联的3 ' -RNA加工。在本项目中，我们旨在确定BRD4控制剪接的分子机制，并表征BRD4与人类细胞剪接体之间的结合界面。为了实现目标，我们将采用高分辨率和定量的多组学方法。

Transcription by RNA polymerase II (Pol II) underlies fundamental cellular processes in eukaryotic cells and is strictly regulated. A major control point occurs during early elongation before Pol II is released into productive elongation. Prior research has linked the BET bromodomain protein family, with its members BRD2, BRD3 and BRD4, with transcription elongation in human cells. Our research has recently provided evidence that the BET bromodomain protein BRD4 underlies the general 5’-elongation checkpoint which helps to assemble a functional Pol II elongation complex and stimulates productive elongation. Transcription elongation is tightly coupled and temporally coordinated with RNA processing to enable RNA maturation while the RNA is being synthesized. We recently uncovered that BRD4 serves as a molecular link between transcription elongation and RNA processing. We found that BRD4 recruits 3’-RNA processing factors to transcriptionally engaged Pol II during the 5’-elongation checkpoint to enable RNA cleavage at the 3’-end of genes (Arnold et al., Mol Cell, 2021). We also found that BRD4 interacts with subunits of the spliceosome and that acute BET protein ablation impacts the splicing pattern at a large set of genes. These findings indicate that BRD4 is implicated in co-transcriptional splicing, similarly to transcription-coupled 3’-RNA processing. In this project, we aim to identify the molecular mechanisms of splicing control by BRD4 and to characterize the binding interface between BRD4 and the spliceosome in human cells. In order to accomplish the objectives, we will apply a high-resolution and quantitative multiomics approach.