An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

Abstract

Formation of the 39 end of RNA polymerase II-transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 39-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 39-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.

Cyclin-dependent Kinase 8 Module Expression Profiling Reveals Requirement of Mediator Subunits 12 and 13 for Transcription of Serpent-dependent Innate Immunity Genes in Drosophila

“…The dependence of gene regulation by Cdk8-CycC on Med12-Med13 noted here and in a genetic study on leg bristles (21) supports the suggested structural hierarchy where Cdk8-CycC is linked to the core Mediator through Med12 and Med13 (17,18). Identification of the dependence of Cdk8-CycC on Med12-Med13 also revealed that these pairs often have opposite transcriptional effects as illustrated in Fig.…”

“…2D. This indicates that opposite regulation by Cdk8-CycC and Med12-Med13 should be considered as a possibility on all Cdk8-CycCregulated genes and functions previously identified to be Med12-Med13-independent (17,18).…”

“…In budding yeast, all four subunits control expression of a common set of genes (14), suggesting that Cdk8 has a major role. In mammalian cells, Cdk8 and Med12 sometimes cooperate on specific genes (15,16), but in other contexts Cdk8 and CycC appear to act independently of Med12, suggesting Mediator-independent functions (17,18). In Drosophila, effects of depletion of Cdk8 and CycC have been compared genome-wide in fat bodies where similar changes were noted (12).…”

Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain That Mediates Activation of the Drosophila Integrator Complex

“…Function-We previously developed a GFP-based reporter system for in vivo monitoring of U7 snRNA 3Ј end formation in Drosophila cells (7,13). The advantage of the U7-GFP reporter as a method of monitoring integrator complex activity is that loss of activity can be readily detected in vivo with high sensitivity via GFP fluorescence.…”

The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

“…Integrator is a metazoan-specific multisubunit, multifunctional protein complex composed of 14 subunits named Int1-Int14 (Integrator subunits) (Baillat et al 2005;Chen et al 2012). Despite the large number of subunits, characteristic structural domains are largely absent, and there are no obvious paralogs of Integrator subunits across animal genomes, except for Int9 and Int11 (Baillat and Wagner 2015).…”