Probing the Role of Integrator in Neuronal Function

项目总结 整合子复合体(INT)是一种与RNA聚合酶II相关的17个亚基的机制 (RNAPII)，并作为关键的转录调节因子发挥作用。3‘-末端的形成是必不可少的。 多种非编码RNA，是启动子近端暂停的RNAPII的广泛负调控因子。 整合子亚基11(INTS11)含有对整合子切割至关重要的RNA内切酶结构域 RNAPII基因座上的新生转录产物，这是转录抑制的一个重要活动。 INTS11基因变异与整合子功能中的基本作用一致，被发现扰乱 并导致一种复杂的神经综合征。同样，人类的基因突变 与INTS11相关的两种非整合子蛋白也会阻碍神经发育，包括 BRAT1，其中变异导致致死性新生儿多灶性癫痫综合征和小脑性共济失调 WDR73，其中变异导致以精神运动为特征的加洛韦-莫瓦特综合征 损伤、低眼压和癫痫发作。 WDR73和BRAT1与INTS11和INTS9相关，但主要是细胞质蛋白 与完整的17亚单位核整合子复合体不同。值得注意的是，人们对此一无所知 这些蛋白结合INTS11或它们在核整合子功能中的作用-特别是在 神经细胞的命运和健康。使用生化和结构方法，我们产生了低温EM INTS11-BRAT1二元络合物和INTS9-INTS11-BRAT1三元络合物的结构。 令人惊讶的是，我们还发现，核积分器复合体内的最佳INTS11功能需要 当缺乏BRAT1或WDR73的细胞积累未切割时，与这些细胞质蛋白的相互作用 积分器基板。总之，这些初步研究产生了一个具有挑衅性的模型 细胞质BRAT1和WDR73需要为INTS11的核活动颁发许可证，这是至关重要的 维持人类正常的神经功能。为了检验这一假设，我们提出了这些具体的 目的：特定目的1.INTS9、INTS11、BRAT1和 WDR73复合体。特定目的2.探讨BRAT1和WDR73在整合子介导中的作用 基因调控。具体目标3.破译含有INTS11的复合体在神经细胞中的作用。

PROJECT SUMMARY The Integrator Complex (INT) is a 17-subunit machinery that associates with RNA polymerase II (RNAPII) and functions as a critical transcription regulator. It is essential for the 3'-end formation of a variety of non-coding RNAs and is a broad negative regulator of promoter-proximally paused RNAPII. Integrator subunit 11 (INTS11) houses the RNA endonuclease domain vital for Integrator to cleave nascent transcripts at all RNAPII loci, which is an important activity for transcriptional repression. Consistent with a fundamental role in Integrator function, INTS11 genetic variants are found to disrupt human development and give rise to a complex neurological syndrome. Similarly, genetic disruptions in two non-Integrator proteins that associate with INTS11 also impede neurodevelopment, including BRAT1, where variants cause a lethal neonatal multi-focus seizure syndrome and cerebellar ataxia, and WDR73, where variants cause Galloway-Mowat syndrome that is characterized by psychomotor impairment, hypotonia, and seizures. WDR73 and BRAT1 associate with INTS11 and INTS9 but are primarily cytoplasmic proteins distinct from the complete 17-subunit nuclear Integrator Complex. Notably, nothing is known about how these proteins bind INTS11 or their role in nuclear Integrator function – especially in the context of neuronal cell fate and fitness. Using biochemical and structural approaches, we generated cryo-EM structures of the INTS11-BRAT1 binary complex and the INTS9-INTS11-BRAT1 ternary complex. Surprisingly, we also find that optimal INTS11 function within the nuclear Integrator Complex requires interaction with these cytoplasmic proteins as cells lacking BRAT1 or WDR73 accumulate uncleaved Integrator substrates. Altogether, these preliminary studies generate a provocative model whereby cytoplasmic BRAT1 and WDR73 are required to ‘license’ INTS11 for its nuclear activity, which is critical to maintaining proper neuronal function in humans. To test this hypothesis, we propose these Specific Aims: Specific Aim 1. Structurally and biochemically characterize the INTS9, INTS11, BRAT1, and WDR73 complexes. Specific Aim 2. Probe the function of BRAT1 and WDR73 in Integrator-mediated gene regulation. Specific Aim 3. Decipher the role of the INTS11-containing complexes in neural cells.