Inositol hexakisphosphate is required for Integrator function

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.

Molecular details of the CPSF73-CPSF100 C-terminal heterodimer and interaction with Symplekin

“…Focussing on the β-barrel formed by the CTD1 of both proteins, we confirm structural similarity to the partial models built for this region from cryo-EM data of the histone pre-mRNA 3' processing machinery (22), as well as IntS11 and IntS9 within Integrator (26)(47) (29). Based on both predicted models and alignments from diverse model organisms (Supplementary Figures S2 and S3), the β-barrel region typically includes loop insertions of varying length, such as the extensive 62 amino-acid loop between strands β2 and β3 in human CPSF100.…”

“…It had been earlier noted that the Ctermini of IntS11 and IntS9 strongly interact (27), and the crystal structure of a IntS11-IntS9 heterodimer of the CTD2 from each protein (23) was already used to help for interpreting the histone pre-mRNA 3'-end processing complex (22). Several recent cryo-EM structures containing Integrator have provided improved resolution data for CTD1 and CTD2 domains of IntS11 and IntS9 (26)(28) (29). Insight into the metazoan CPSF is also provided by structural comparison to the yeast Cleavage and Polyadenylation Factor (CPF) nuclease module which contains Ysh1 and Cft2 (orthologues to CPSF73 and CPSF100), but at present the C-termini of these two proteins have not been observable by structural methods (30) (31).…”

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3′ processing machinery

“…First, Compound 2 activity may be similar to that of a chemical that is more universally found in cells. A number of small molecules, including inositol hexakisphosphate, can bind to and modulate the activities of molecular machinery in the gene expression pathway, such as the spliceosome 31 , the Integrator complex 32 , and the mRNA export factors 33 . It is possible that a naturally occurring Compound 2-like small molecule can inhibit pre-mRNA 3¢ processing and many PASs evolved to overcome such inhibition.…”

SUMO conjugation regulates the activity of the Integrator complex

“…To study the role of IP 6 binding, the positively charged Lys462 was replaced by a negative residue (Glu) within the intS11 subunit. This change impaired the interaction with other subunits of the Integrator complex, affecting its function ( 64 ). Nevertheless, here we show that even without altering the charge of residue 462 (replacing Lys by Arg) or by abrogating the SUMO consensus motif surrounding Lys462, SUMOylation disruption in that position shifts the subcellular localization of INTS11.…”