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Analyses of the essential C82 subunit uncover some differences in RNA polymerase III transcription between Trypanosoma brucei and Leishmania major

ABSTRACT: The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the four distinctive winged-helix domains and the coiled-coil motif are present in C82 in these microorganisms, despite the observation of certain unique traits. Also, their projected three-dimensional structure is comparable to that of the human orthologue. C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. In T. brucei, knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of the pathogen. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Trypanosoma Brucei Leishmania Major Strain Friedlin

TISSUE(S): Cell Culture

SUBMITTER: Ebbing de Jong

LAB HEAD: Santiago Martínez-Calvillo

PROVIDER: PXD051017 | Pride | 2024-11-13

REPOSITORIES: Pride

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			Action	DRS
	20201211_SC_WT.mgf	Mgf
	20201211_SC_WT.mzML	Mzml
	20201211_SC_WT.mzid.gz	Mzid
	20201211_SC_WT.raw	Raw
	20210602_SC_TB_C82_1.mgf	Mgf

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Project description:RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a single putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131 and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major.

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