Inositol hexakisphosphate is required for Integrator function

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.