A TFIIH-Associated Mediator Head Is a Basal Factor of Small Nuclear Spliced Leader RNA Gene Transcription in Early-Diverged Trypanosomes

Abstract

Genome annotation suggested that early-diverged kinetoplastids possess a reduced set of basal transcription factors. More recent work, however, on the lethal parasite Trypanosoma brucei identified extremely divergent orthologs of TBP, TFIIA, TFIIB, and TFIIH which, together with the small nuclear RNA-activating protein complex, form a transcription preinitiation complex (PIC) at the spliced leader (SL) RNA gene (SLRNA) promoter. The SL RNA is a small nuclear RNA and a trans splicing substrate for the maturation of all pre-mRNAs which is metabolized continuously to sustain gene expression. Here, we identified and biochemically characterized a novel TFIIH-associated protein complex in T. brucei (Med-T) consisting of nine subunits whose amino acid sequences are conserved only among kinetoplastid organisms. Functional analyses in vivo and in vitro demonstrated that the complex is essential for cell viability, SLRNA transcription, and PIC integrity. Molecular structure analysis of purified Med-T and Med-T/TFIIH complexes by electron microscopy revealed that Med-T corresponds to the mediator head module of higher eukaryotes. These data therefore show that mediator is a basal factor for small nuclear SL RNA gene transcription in trypanosomes and that the basal transcription function of mediator head is a characteristic feature of eukaryotes which developed early in their evolution.

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

“…TFIIH/CDK7-mediated CTD phosphorylation disrupts this interaction, presumably facilitating promoter escape of the polymerase (56). Trypanosomes do possess a mediator complex that is essential for SLRNA transcription, interacts with TFIIH, and is required for the formation of a transcription preinitiation complex at the SLRNA promoter (18). However, whereas the eukaryotic mediator consists of head, middle, tail, and CDK8 modules, electron microscopic structure analysis of the trypanosome complex indicated that it is confined to the head module (18).…”

“…Thus, the absence of a TFIIH kinase (17) and the presence of a minimal mediator complex suggest that, in trypanosomes, a mediator-CTD interaction does not occur and a release of RNA Pol II from the mediator is not a prerequisite for promoter escape of the enzyme. Accordingly, tandem affinity purification of the trypanosome mediator did not copurify detectable amounts of RNA Pol II subunits (18), and vice versa (20,21).…”

“…Trypanosomes do possess a mediator complex that is essential for SLRNA transcription, interacts with TFIIH, and is required for the formation of a transcription preinitiation complex at the SLRNA promoter (18). However, whereas the eukaryotic mediator consists of head, middle, tail, and CDK8 modules, electron microscopic structure analysis of the trypanosome complex indicated that it is confined to the head module (18). Thus, the absence of a TFIIH kinase (17) and the presence of a minimal mediator complex suggest that, in trypanosomes, a mediator-CTD interaction does not occur and a release of RNA Pol II from the mediator is not a prerequisite for promoter escape of the enzyme.…”

“…Trypanosome RNA Pol II transcribes the protein coding gene arrays and the ϳ100 tandem copies of the SL RNA gene (SLRNA) on chromosome 9, whereas other snRNAs in trypanosomes are synthesized by RNA Pol III. SLRNA transcription is monocistronic, initiates at a distinct initiation site, and requires the assembly of a conventional, albeit extremely divergent, preinitiation complex (16)(17)(18). The T. brucei genome encodes a full set of RNA Pol II subunits, termed RPB1 to RPB12 (19), and biochemical characterization of the enzyme complex identified all subunits except RPB10 (20)(21)(22).…”

Structural flexibility and functional interaction of mediator Cdk8 module

“…Not only acquiring the structural information, single particle EM is ideally suited to characterize the conformation of Mediator under different conditions and illuminate how it is affected by interaction with various factors. Understanding the interaction of Mediator with the general RNAPII machinery has derived from EM structures of the Mediator-RNAPII complex (Davis et al, 2002;Bernecky et al, 2011), Mediator-TFIIH complex (Lee et al, 2010), Mediator Head-TBP , and Mediator Head-RNAPII (Cai et al, , 2012.…”