A combinatorial approach to uncover a novel Integrator subunit [RNA-seq]

The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Recent structural, biochemical, and functional evidence suggest that Integrator is composed of 14 subunits, assembling, and operating in a modular fashion. We employed proteomics and machine-learning (alphafold2) approaches to discover a novel Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS10-INTS13-INTS14 module and interfaces with the Int-PP2A phosphatase module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes in human cell lines. C7orf26 (INTS15) was knocked down in HeLa cells, serum starved, and transcription was induced through the addition of Epidermal Growth Factor (EGF) to the media. After 15 minutes, the cells were collected for total RNA-seq.

整合子复合物（Integrator complex）是后生动物中最难以捉摸的转录调控因子之一，被认为会在转录起始后被招募，以协助建立并调控处于暂停状态的RNA聚合酶II（RNAPII）。近期的结构生物学、生物化学与功能研究证据表明，整合子复合物由14个亚基组成，并以模块化方式完成组装并发挥功能。本研究通过蛋白质组学与机器学习（AlphaFold2）技术手段，发现了一个全新的整合子复合物亚基INTS15。研究证实，INTS15主要与INTS10-INTS13-INTS14模块组装结合，并与Int-PP2A磷酸酶模块形成相互作用界面。功能基因组学分析进一步揭示，INTS15在人类细胞系的部分基因位点中参与调控RNA聚合酶II的暂停过程。本研究在HeLa细胞中敲低了C7orf26（INTS15）的表达，对细胞进行血清饥饿处理，并通过向培养基中添加表皮生长因子（EGF）诱导转录。处理15分钟后，收集细胞并提取总RNA进行RNA测序（RNA-seq）。