Mutual Exclusivity of MED12/MED12L, MED13/13L, and CDK8/19 Paralogs Revealed within the CDK-Mediator Kinase Module

The macromolecular complex Mediator plays important roles in regulation of RNA Polymerase II (RNAPII) activity by DNA-binding proteins, non-coding RNAs, and chromatin. Structural and biochemical studies have shown that human Mediator exists in two forms; a core complex of 26 proteins, termed Mediator, and a larger complex containing the CDK8 kinase module, termed CDK8-Mediator. Interestingly, 3 subunits of the kinase module have undergone independent gene duplications in vertebrates to generate the paralog pairs MED12/MED12L, MED13/ MED13L, and CDK8/CDK19. Each has been shown to interact with Mediator, yet clearly defining the composition of CDK kinase module has been challenging due to the large size (~600 kD), the similarities between paralogs, and potential combinatorial nature of complexes. In this study, we performed a systematic proteomic analysis using HaloTag technology to isolate each kinase module member (MED12, MED12L, MED13, MED13L, CDK8, CDK19, and Cyclin C) and their interacting partners from HEK293T cells. Using LC-MS/MS we were able to differentiate paralogs within samples by specific analysis of unique peptides. We found that the paralogs assemble into CDKMediator in a mutually exclusive fashion, thus allowing for up to eight different assemblies of the CDK module, with potential for functional specialization of Mediator complexes. Interestingly, we found thatMED13L complexes carry the MED26 subunit, which has been shown to be either absent or in very low abundance in CDK-Mediator. This unique variant of the Mediator complex may reconcile recent observations demonstrating roles for MED26 and CDK8 in positive control of RNAPII elongation. Together these data expand the understanding of CDK-Mediator complexes and composition.