Inositol hexakisphosphate is a critical regulator of Integrator assembly and function

Abstract

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 A resolution, unexpectedly revealed the stable association of an inositol hexakisphosphate (IP6) molecule. The binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 A away from the IntS11 active site and generally conserved in other ICMs. IP6 binding is also confirmed in human ICM. Mutations of residues in this binding site or disruption of IP6 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 IP6 is a critical regulator of Integrator assembly and function in Drosophila, humans, and likely other organisms.

Emerging insights into the function and structure of the Integrator complex

“…Among all Integrator subcomplexes, the cleavage module is structurally best characterized [ 11 , 18 , 20 , 68 ]. While most Integrator subunits are mainly α-helical with very few predictable domain features, INTS9/INTS11 adopt MBL/β-CASP folds with remarkable similarity to their corresponding orthologs CPSF100/CPSF73 [ 69 ].…”

“…The C-terminal region of INTS4 forms a β-sandwich, which sits on top of the β-CASP domain of INTS9. Together, INTS4/9/11 forms a highly positively charged cavity, which serves as a conserved binding site for inositol hexaphosphate (IP6) [ 68 ]. Mutations close to the IP6-binding site cause nuclease malfunctions in reporter assays; however, it is unknown whether IP6 contributes to Integrator activity or has only a scaffolding function [ 18 , 68 ].…”