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. 2024 Dec;108(1):109.
doi: 10.1007/s00253-023-12903-8. Epub 2024 Jan 10.

Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites

Fabiola Mondragón-Rosas  1 , Luis E Florencio-Martínez  1 , Gino S Villa-Delavequia  1 , Rebeca G Manning-Cela  2 , Julio C Carrero  3 , Tomás Nepomuceno-Mejía  1 , Santiago Martínez-Calvillo  4
Affiliations

Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites

Fabiola Mondragón-Rosas et al. Appl Microbiol Biotechnol. 2024 Dec.

Abstract

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 published 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 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. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.

Keywords: 5S rRNA; Leishmania major; RNAP III transcription; TFIIIC; Tau95; Trypanosoma brucei; tRNA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Sequence analysis and predicted three-dimensional structure of Tau95 in trypanosomatids. a Protein sequence alignment of the dimerization and DNA binding domains of Tau95 from T. brucei (Tb, Tb927.10.980), L. major (Lm, LmjF.21.1100), and S. pombe (Sp, NP_593297). Conserved residues are denoted by black shading, conserved substitutions are indicated by dark-gray shading, and semiconserved substitutions are denoted by light-gray shading, according to the Clustal Ω program. Predicted secondary structure elements are shown for T. brucei (above the sequence) and for S. pombe (below the sequence). The α-helices are indicated with rectangles and the β-strands with arrows. b Predicted three-dimensional structure of the dimerization and DNA binding domains of TbTau95 by homology modeling using the crystal structure of S. pombe Tau95 as a template. The structures are shown in the same colors presented in panel a. The quality of the models was estimated with Mod Eval server (https://modbase.compbio.ucsf.edu/evaluation/), showing a score of 0.70. The location of the winged helix (WH) and winged helix interacting (WHI) domains is indicated
Fig. 2
Fig. 2
Nuclear localization of TbTau95. a Western blot analysis with total protein from wild-type (WT) parasites and transgenic cells that express the TbTau95-PTP protein using an anti-Prot C monoclonal antibody. β-tubulin was used as a loading control. b The location of TbTau95-PTP was determined by indirect immunofluorescence assays using anti-Prot C monoclonal antibody and an Alexa-Fluor 488 conjugated secondary antibody (Life Technologies Corporation, Grand Island, NY, USA). Nucleus (N) and kinetoplast (K) were stained with DAPI. Size bars represent 5 μm
Fig. 3
Fig. 3
LmTau95 is a nuclear protein. a Western blot analysis with cells that express the recombinant protein LmTau95-PTP and wild-type (WT) cells. Membranes were incubated with an antibody against Prot C and an anti-β-tubulin antibody (loading control). b Indirect immunofluorescence experiments to determine the subcellular localization of LmTau95-PTP using an anti-Prot C antibody. As a nucleolar marker, an anti-LmNop56 antibody was used. Parasites were then treated with a mixture of secondary anti-rabbit antibody conjugated with Alexa-Fluor 488 and anti-mouse antibody conjugated with Alexa Fluor 568 (Life Technologies Corporation, Grand Island, NY, USA). Nucleus (N) and kinetoplast (K) were stained with DAPI. Size bars represent 5 μm
Fig. 4
Fig. 4
Attempt to knockdown TbTau95 by RNAi. a Growth curve of a clonal cell line obtained with the pZJM vector under non-induced (Dox−) and doxycycline-induced (Dox+) conditions. Cells were counted daily and diluted to a density of 2 × 106 cells/ml. The values represent the cumulative cell density multiplied by the dilution factor. Data points reflect the means of triplicate experiments. Standard deviation bars are shown. b Northern blot analysis of TbTau95 mRNA in non-induced cells (Dox−), and cells induced for 24, 48, 72, and 96 h. RNA from wild-type (WT) cells was also analyzed. As a loading control, the filter was stripped and re-hybridized with an α-tubulin probe. c RT-qPCR experiments to determine the relative abundance of TbTau95 mRNA in non-induced cells (0 h) and cells induced for 24 and 96 h. d Western blot analysis of TbTau95 protein in non-induced cells (0 h), and cells induced for 24, 48, 72, and 96 h using a specific anti-TbTau95 polyclonal antibody at 1:5000 dilution. The bands shown here and from two independent experiments were quantified and plotted, considering as 100% the protein level obtained in the non-induced culture. Values represent means of the three experiments. Error bars indicate standard errors. TbTau95 protein levels were normalized to the level of the β-tubulin protein (loading control). e RT-qPCR assays to determine the relative abundance several transcripts in non-induced cells (0 h) and cells induced for 96 h. The transcripts analyzed were tRNA-Ala, tRNA-Arg, U2 snRNA, 5S rRNA, and the mRNAs from TFIIB and Procyclin. Error bars indicate standard errors
Fig. 5
Fig. 5
Chromatin immunoprecipitation analysis of TbTau95. a Schematic representation of the genes and promoter regions that were investigated. Genomic regions transcribed by RNAP III, RNAP II, and RNAP I are shown in orange, blue and green, respectively. b A ChIP grade anti-Prot A antibody was used to precipitate chromatin from the cell line that expresses the TbTau95-PTP protein. Precipitated DNA was examined by qPCR. The results from three independent ChIP experiments, each analyzed by two qPCR reactions, are shown. Error bars indicate standard deviations. Results are presented as fold enrichment over negative control precipitations
Fig. 6
Fig. 6
Tandem affinity purifications with parasites expressing Tau95-PTP recombinant proteins. SDS-PAGE of proteins copurified with TbTau95-PTP (a) and LmTau95-PTP (b). The asterisks indicate the recombinant proteins. Control experiments with wild-type (WT) T. brucei (a) and L. major (b) cells are also presented. Proteins were analyzed in 4–15% Mini- PROTEAN Precast Protein Gels (Bio-Rad, Hercules, CA, USA) stained with SYPRO Ruby (Invitrogen, Carlsbad, CA, USA)
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References

    1. Acker J, Conesa C, Lefebvre O. Yeast RNA polymerase III transcription factors and effectors. Biochim Biophys Acta. 2013;1829(3–4):283–295. doi: 10.1016/j.bbagrm.2012.10.002. - DOI - PubMed
    1. Alsford S, Turner DJ, Obado SO, Sanchez-Flores A, Glover L, Berriman M, Hertz-Fowler C, Horn D. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011;21(6):915–924. doi: 10.1101/gr.115089.110. - DOI - PMC - PubMed
    1. Bhalla P, Shukla A, Vernekar DV, Arimbasseri AG, Sandhu KS, Bhargava P. Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes. Sci Rep. 2019;9(1):12892. doi: 10.1038/s41598-019-49316-5. - DOI - PMC - PubMed
    1. Bhalla P, Vernekar DV, Gilquin B, Couté Y, Bhargava P. Interactome of the yeast RNA polymerase III transcription machinery constitutes several chromatin modifiers and regulators of the genes transcribed by RNA polymerase II. Gene. 2019;702:205–214. doi: 10.1016/j.gene.2018.12.037. - DOI - PubMed
    1. Billington K, Halliday C, Madden R, Dyer P, Barker AR, Moreira-Leite FF, Carrington M, Vaughan S, Hertz-Fowler C, Dean S, Sunter JD, Wheeler RJ, Gull K. Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei. Nat Microbiol. 2023;8(3):533–547. doi: 10.1038/s41564-022-01295-6. - DOI - PMC - PubMed

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