The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs.