Abstract
Here we present the most comprehensive analysis of the yeast Mediator complex interactome to date. Particularly gentle cell lysis and co-immunopurification conditions allowed us to preserve even transient protein-protein interactions and to comprehensively probe the molecular environment of the Mediator complex in the cell. Metabolic 15N-labeling thereby enabled stringent discrimination between bona fide interaction partners and nonspecifically captured proteins. Our data indicates a functional role for Mediator beyond transcription initiation. We identified a large number of Mediator-interacting proteins and protein complexes, such as RNA polymerase II, general transcription factors, a large number of transcriptional activators, the SAGA complex, chromatin remodeling complexes, histone chaperones, highly acetylated histones, as well as proteins playing a role in co-transcriptional processes, such as splicing, mRNA decapping and mRNA decay. Moreover, our data provides clear evidence, that the Mediator complex interacts not only with RNA polymerase II, but also with RNA polymerases I and III, and indicates a functional role of the Mediator complex in rRNA processing and ribosome biogenesis.
Highlights
The Mediator complex is an essential coactivator of eukaryotic transcription
In addition to activators and components of the preinitiation complex (PIC), we identified a number of interactors that are involved in Polymerase II (Pol II) elongation and in co-transcriptional processes
The conditions applied in our study allowed us to identify an extraordinary large number of specific interactors, providing a much more comprehensive picture of Mediator’s cellular environment
Summary
The Mediator complex is an essential coactivator of eukaryotic transcription. Its major function is to communicate regulatory signals from gene-specific transcription factors upstream of the transcription start site to RNA Polymerase II (Pol II) and to promote activator-dependent assembly and stabilization of the preinitiation complex (PIC)[1,2,3]. There is growing evidence that the Mediator complex is involved in post-initiation stages of Pol II transcription[5,6]. Mass spectrometry contributed in various ways to our current knowledge about the Mediator complex. A number of interaction partners of the Mediator complex were identified by large-scale mass spectrometry-based interactome analyses in yeast[21,22] as well as in human cell lines[23,24]. The overall number of Mediator interaction partners identified from these studies is limited. We think this is mainly due to the cell lysis and CoIP conditions applied in these studies, which are suboptimal in preserving transient interactions. In order to obtain a more complete picture of the various cellular functions of the Mediator complex and in order to identify potential regulators of the Mediator complex we performed a comprehensive analysis of the yeast Mediator complex interactome
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