Abstract
Integrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3'-end processing and transcription attenuation of a broad collection of mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 Integrator cleavage module (ICM). Here we report a cryo-EM structure of the Drosophila ICM, at 2.74 Å resolution, revealing stable association of an inositol hexakisphosphate (IP6) molecule. The IP6 binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 Å away from the IntS11 active site and generally conserved in other ICMs. We also confirmed IP6 association with the same site in human ICM. IP6 binding is not detected in ICM samples harboring mutations in this binding site. Such mutations or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3'-end processing and mRNA transcription attenuation. Our structural and functional studies reveal that IP6 is required for Integrator function in Drosophila, humans, and likely other organisms.