Identification and structural basis of a potential licensing factor of the Integrator cleavage module

Abstract: Transcription is one of the first steps in gene expression, and the many protein factors involved must be tightly regulated to correctly carry out cellular processes. The Integrator complex, a 15-subunit metazoan- specific complex, was initially discovered to be crucial for the proper transcription of certain non-coding RNAs. Recently, it has been found that Integrator is a broad transcriptional regulator with roles at many protein-coding and non-coding genes. Integrator acts as a negative regulator of RNA Polymerase II (RNAPII) transcription through promoter-proximal termination and can alter its functionality in response to stress, such as DNA damage or reactive oxygen species. Despite its critical role in fine-tuning transcriptional regulation, it is not yet understood how Integrator is assembled and regulated, presenting a knowledge gap in the field. Our preliminary experiments strikingly revealed that a key factor involved in DNA damage repair (DDR) associates with a global regulator of RNAPII activity, thus providing a previously unknown connection between these two processes. Specifically, the BRCA1-ATM activator 1 (BRAT1) associates with Integrator subunit 11 (INTS11), the endonuclease-containing subunit within the Integrator complex. BRAT1 is a cytoplasmic protein that re-localizes to the nucleus upon DNA damage to act as a scaffold between the site of damage and repair proteins. Our cryo-EM analysis of the INTS11-BRAT1 heterodimer reveals that BRAT1 forms a circular structure around INTS11 and uses three conserved C-terminal residues to bind to the INTS11 active site, preventing its cleavage of RNA. Additionally, the cryo-EM analysis showed that INTS9 can bind to form an INTS11-INTS9- BRAT1 heterotrimer, suggesting interplay between BRAT1 as an inhibitor of INTS11 and other members of the Integrator complex. The goal of this proposal is to examine the function and mechanism of action of these two unique complexes. To examine the function, our lab generated BRAT1 null cell lines and a rapid depletion system using the auxin-inducible degron (AID) tag to allow quick depletion of INTS11. Initial studies of these lines show that BRAT1 loss results in an INTS11 depletion phenotype, presenting upregulation of genes negatively regulated by Integrator. Overall, these data present a model where BRAT1 could regulate Integrator activity through its association with INTS9/11. The hypothesis is that BRAT1 acts as a licensing factor, facilitating the assembly of the Integrator complex and providing precise regulation, which is especially important during cellular stress such as DNA damage. To test this hypothesis, three aims are proposed. First, the interaction between BRAT1 and INTS9/11 will be biochemically defined to further probe the structure. Next, the impact of BRAT1 on the canonical Integrator complex function will be determined. Finally, the impact of INTS11-INTS9-BRAT1 during DNA damage repair will be elucidated. Successful completion of these aims will provide a more thorough understanding of the regulation and assembly of the Integrator complex.