Probing the Role of Integrator in Neuronal Function

项目摘要 整合子复合物（INT）是一个17亚基的机器，与RNA聚合酶II相关 （RNAPII）并作为关键的转录调节因子发挥作用。它是一个重要的3 '-末端形成的一个 是启动子近端暂停的RNAPII的广泛负调节因子。 整合子亚基11（INTS 11）包含对整合子切割至关重要的RNA内切酶结构域 在所有RNAPII基因座的新生转录物，这是转录抑制的重要活动。 与整合子功能中的基本作用一致，发现INTS 11遗传变异破坏了 人类发展和引起一种复杂的神经系统综合症。同样地， 与INTS 11相关的两种非整合蛋白也阻碍神经发育，包括 BRAT 1，其中变体引起致死性新生儿多灶性癫痫综合征和小脑共济失调，以及 WDR 73，其中变体引起以精神错乱为特征的Galloway-Mowat综合征 损伤、张力减退和癫痫发作。 WDR 73和BRAT 1与INTS 11和INTS 9相关，但主要是细胞质蛋白 与完整的17-亚基核整合子复合物不同。值得注意的是，没有人知道 这些蛋白结合INTS 11或它们在核整合子功能中的作用-特别是在 神经元细胞的命运和适应性。使用生物化学和结构方法，我们产生了冷冻EM INTS 11-BRAT 1二元复合物和INTS 9-INTS 11-BRAT 1三元复合物的结构。 令人惊讶的是，我们还发现，核整合复合物中的最佳INTS 11功能需要 当缺乏BRAT 1或WDR 73的细胞积累未切割的蛋白质时， 积分器衬底。总之，这些初步研究产生了一个挑衅性的模型， 细胞质BRAT 1和WDR 73需要“许可”INTS 11的核活性，这是至关重要的 来维持人类正常的神经功能为了验证这一假设，我们提出了这些具体的 目标：具体目标1。对INTS 9、INTS 11、BRAT 1和 WDR 73复合物。具体目标2。BRAT 1和WDR 73在整合子介导的细胞凋亡中的作用 基因调控具体目标3。解密INTS 11复合物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.