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. 2012 Dec;18(12):2148-56.
doi: 10.1261/rna.035725.112. Epub 2012 Oct 24.

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

Jiandong Chen  1 , Nader Ezzeddine, Bernhard Waltenspiel, Todd R Albrecht, William D Warren, William F Marzluff, Eric J Wagner
Affiliations

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

Jiandong Chen et al. RNA. 2012 Dec.

Abstract

Formation of the 3' 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 3'-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 3'-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.

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Figures

FIGURE 1.
FIGURE 1.
Using the U7-GFP reporter to conduct a genome-wide RNAi screen. (A) Schematic of the U7-GFP reporter and the results from transient transfection of the reporter into S2 cells treated with dsRNA targeting PTB (−control) or IntS12 (+control). (Left) Fluorescence and bright-field images; (right) Western blot analysis. (B) Bar graph representing quantification of screen results for plates 25 and 54. Insets are taken from the acquired image collection. (C) Pie graph representing results of RNAi screen categorically. (D) Trimmed-down list of non-Integrator proteins that reproducibly scored as strong or modest by secondary screening.
FIGURE 2.
FIGURE 2.
Validation of Asunder, CG4785, CycC, and Cdk8 as required for snRNA 3′-end formation. (A) Images from S2 cells transfected with the U7-GFP reporter after dsRNA treatment targeting Asu or CG4785. (B) Western blot analysis of cell lysates from A. (C) qRT-PCR analysis specific for misprocessed endogenous snRNA from knockdown cells. (D) Western blot analysis of S2 cells treated with dsRNAs targeting CycC or Cdk8 followed by transient transfection with reporters measuring snRNA or histone mRNA 3′-end formation. (E) Western blot analysis of cells cotransfected with U7-GFP reporter and myc-tagged Cdk8 that is wild type or catalytically inactive.
FIGURE 3.
FIGURE 3.
Cyclin C and Cdk8 are not involved in regulating Integrator expression. (A) Western blot analysis of protein levels of Integrator subunits after treating cells with dsRNAs targeting LacZ, CycC, or Cdk8. Cells were also transfected with the U7-GFP reporter to monitor snRNA misprocessing as a functional readout of knockdown of CycC/Cdk8. (B) Quantitative RT-PCR analysis using SYBR Green staining of PCR products. RNA was isolated from S2 cells, treated with the three dsRNAs described in A, and subjected to real-time PCR analysis using amplicons specific to Integrator subunits not tested by Western blot analysis in A. All measurements were normalized to RpS17 signals as a housekeeping internal control. (C) Ethidium bromide staining of agarose gel electrophoresis of the amplicons amplified in B.
FIGURE 4.
FIGURE 4.
Asunder and CG4785 biochemically associate with the fly Integrator complex. (A) Western blot analysis of Flag-tagged Asunder or CG4785 expression in stable S2 cell lines. (B) IF analysis of stable S2 cell lines expressing Flag-tagged Asunder or CG4785. (C) Western blot analysis of Flag immunoprecipitates from cell lines described in A. Input lanes represent 5% of input, and IP represents 50% of the immunoprecipitate. (D) Directed yeast two-hybrid using fly Integrator subunits and CG4785 demonstrating an interaction with IntS10.
FIGURE 5.
FIGURE 5.
CycC/Cdk8 function in snRNA 3′-end formation independent of Mediators 12/13 and are associated with Integrator subunits. (A) Western blot analysis of lysates from S2 cells treated with dsRNAs targeting members of the Drosophila Mediator Cdk8 module and transfected with the U7-GFP reporter. (B) Western blot analysis of lysates from HeLa cells treated with siRNA to CycC or CDK8 followed by transfection with a human version of the U7-GFP reporter. (C) Immunoprecipitations using αFlag-agarose to detect interactions between CycC/Cdk8 and Integrators 1 and 9. Input lanes represent 2% of input, and IP lanes represent 50% of immunoprecipitate. (D) Western blot analysis of immunoprecipitations using anti-IntS12 antibodies.
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