Inositol hexakisphosphate is a critical regulator of Integrator assembly and function

Integrator has critical roles in noncoding RNA 3′-end processing as well as transcription attenuation of selected mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 complex (Integrator cleavage module, ICM). Our structure of the Drosophila ICM, determined by cryo-electron microscopy at 2.74 Å resolution, unexpectedly revealed the stable association of an inositol hexakisphosphate (IP 6 ) molecule. The 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. IP 6 binding is also confirmed in human ICM. Mutations of residues in this binding site or disruption of IP 6 biosynthesis significantly reduced Integrator assembly and activity in snRNA 3′-end processing. Our structural and functional studies reveal that Integrator is subject to intricate cellular control and IP 6 is a critical regulator of Integrator assembly and function in Drosophila , humans, and likely other organisms.