ChIP of K4Me3 in Ctr condition and Mago knockdown [Promoter-proximal pausing mediated by the exon junction complex regulates splicing]

Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition. Overall design: K4Me3 ChIP in control and Mago depleted S2R+ cells, in duplicatesRNA聚合酶II（RNA polymerase II，Pol II）的启动子近端暂停是后生动物中广泛存在的一类转录调控步骤。本研究发现核外显子连接复合物（pre-EJC）是该过程的关键且保守的调控因子。敲低pre-EJC亚基会导致Pol II暂停的全局水平下降，并使转录提前进入延伸阶段。该效应至少部分通过在启动子区域非经典招募pre-EJC组分得以实现。启动子区域无法招募pre-EJC会导致正性转录延伸复合物（P-TEFb）结合水平升高，并增强Pol II的释放。值得注意的是，恢复Pol II暂停水平足以挽救外显子跳跃现象，以及体内敲低pre-EJC组分所引发的感光细胞分化缺陷。我们提出，pre-EJC可作为早期转录检查点，阻止转录提前进入延伸阶段，从而确保招募外显子定义过程所需的RNA加工组分。整体实验设计：在对照组与Mago敲低的S2R+细胞中开展K4Me3染色质免疫沉淀（ChIP）实验，设置生物学重复。