Abstract
Mediator complexes are large multiprotein assemblies that function in the regulation of eukaryotic gene transcription. In yeast, certain mediator subunits appear to comprise a subcomplex that acts in the regulation of a specific subset of genes. We investigated in a metazoan, Caenorhabditis elegans, the roles and interactions of two of those subunits, CeTRAP240/let-19 and CeTRAP230/dpy-22. We found that CeTRAP240/let-19 contains four domains that are conserved in the human TRAP240 protein and that one of those domains displays intrinsic transcriptional repression activity. Using RNA interference, we found that reduced expression of CeTRAP240/let-19 displayed a high penetrance of embryonic lethality in F1 progeny; animals that escaped embryonic arrest showed mutant phenotypes such as burst vulva and molting defects. CeTRAP240/let-19 appeared to affect specific genes, as CeTRAP240/let-19(RNAi) led to selectively reduced expression of a subset of reporter genes examined. Genetic experiments supported the view that CeTRAP240/let-19 and CeTRAP230/dpy-22, like their Drosophila and yeast counterparts, can operate on common pathways. Thus, a male tail phenotype caused by the pal-1(e2091) mutation was suppressed not only by CeTRAP230/dpy-22 mutants, as reported previously, but also by reduced expression of CeTRAP240/let-19. Additionally, CeTRAP240/let-19(RNAi) in a CeTRAP230/dpy-22 mutant background produced a strong synthetic lethal phenotype. Overall, our results establish specific roles of CeTRAP240/let-19 in C. elegans embryonic development and a functional interaction between CeTRAP240/let-19 and CeTRAP230/dpy-22. Interestingly, whereas this interaction has been conserved from yeast to mammals, the subcomplex modulates metazoan-specific genetic pathways, likely in addition to those also controlled in yeast.
Highlights
The Mediator complex is a multiprotein assembly that participates in both positive and negative regulation of transcription by RNA polymerase II in eukaryotes; it was first identified in Saccharomyces cerevisiae using biochemical and genetic approaches (1– 4)
We demonstrated important roles for CeTRAP240/let-19 in C. elegans embryonic development and showed that it is likely involved in regulating the expression of a specific subset of genes
The reduced expression of CeTRAP240/let-19 by RNA interference (RNAi) is unlikely to disrupt the integrity of the Mediator complex because in vitro studies of mammalian and yeast mediator complexes have demonstrated the presence of complexes lacking cyclin-dependent kinase 8-cyline c-TRAP230-TRAP240
Summary
Strains—C. elegans (N2 wild type and derived mutant strains) was grown at room temperature. For bacterial feeding RNAi experiments, cDNA corresponding to the nucleotide sequence 3541–5386 of CeTRAP240 cDNA was amplified by PCR and subcloned into the KpnI and PstI sites of plasmid pAD12 (provided by Andy Dillon, University of California, San Francisco), which contains two T7 promoters in inverted orientation This construct was transformed into an IPTG-inducible Escherichia coli strain, HT115, which lacks double-strand-specific RNase III. This PCR fragment was digested by BamHI and XbaI and subcloned into pSG424 plasmid (39). Transfection of A549 lung adenocarcinoma cells employed LipofectAMINE 2000 (Invitrogen) according to the technical manual; cells were harvested 24 h post-transfection
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