Integrator subunit 4 is a ‘Symplekin-like’ scaffold that associates with INTS9/11 to form the Integrator cleavage module

Abstract

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3′-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a β-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3′-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator ‘cleavage module’ responsible for its endonucleolytic activity.

The Integrator complex cleaves nascent mRNAs to attenuate transcription

“…5C; Supplemental Fig. S9; Albrecht et al 2018). Moreover, analogous to our prior results with MtnA (Fig.…”

“…1E; Supplemental Fig. S3A; Ezzeddine et al 2011;Albrecht et al 2018). Nevertheless, because mature snRNAs have long half-lives (Fury and Zieve 1996), their levels only marginally decreased over the 72 h time course of the experiment (Supplemental Fig.…”

INTS10–INTS13–INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module

“…Instead of existing as a monolithic holo-complex, evidence from several studies suggests that INT might assemble in a stepwise manner from separate modules 2 , 3 : For example, the catalytic heterodimer INTS9–INTS11 was shown to copurify with INTS4 from nuclear cell extracts in a lower molecular weight peak distinct from the holo-complex, suggesting that these three subunits form the cleavage module of INT 4 , 34 . Consistently, targeted ChIP analyses of several INTS on U2 snRNA suggest that INTS11 joins the core of the complex only towards the 3′-end 20 .…”

Attenuation of Eukaryotic Protein-Coding Gene Expression via Premature Transcription Termination

“…The IntS4, IntS9, and IntS11 proteins interact with one another (Fig. 1C; Wu et al 2017;Albrecht et al 2018) and, like the Cpsf73, Cpsf100, Symplekin complex ( Fig. 1A; Sullivan et al 2009), can catalyze cleavage of RNA.…”

“…IntS4, IntS9, and IntS11, which comprise the Integrator cleavage module (Fig. 1C; Albrecht et al 2018), are most important for snRNA processing, whereas the noncatalytic subunits play only minor roles (Fig. 3).…”