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.