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. 2021 Feb 16;12(2):280.
doi: 10.3390/genes12020280.

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

Luis E Florencio-Martínez  1 , Andrés Cano-Santiago  1 , Fabiola Mondragón-Rosas  1 , Maricarmen Gómez-García  1 , Carlos Flores-Pérez  1 , Fiordaliso C Román-Carraro  1 , Luis A Barocio-Rodríguez  1 , Rebeca G Manning-Cela  2 , Tomás Nepomuceno-Mejía  1 , Santiago Martínez-Calvillo  1
Affiliations

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

Luis E Florencio-Martínez et al. Genes (Basel). .

Abstract

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

Keywords: 5S rRNA; Leishmania; Pol III transcription; RNA polymerases; TFIIIB; tRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sequence and predicted structure analyses of LmBrf1. (A) Sequence alignment of the N-terminal region and homology blocks I–III of TFIIB-related factor 1 (Brf1) from Leishmania major (Lm, LmjF.25.0440) and Saccharomyces cerevisiae (Sc, CAA68968.1). Please note that the last two lines of the alignment (Homology Block II and Homology Block III) are not contiguous with each other and with the rest of the alignment. Conservation is denoted by black shading, conserved substitutions are indicated by dark-gray shading, and semiconserved substitutions are denoted by light-gray shading, according to the ClustalΩ program. The zinc ribbon, both cyclin repeats, and the Brf1 homology blocks I, II, and III are indicated. Predicted secondary structure elements are shown for L. major (above the sequence) and for S. cerevisiae (below the sequence). The β-strands are denoted by yellow arrows, and α-helices by rounded rectangles. The five α-helices of the first and the second cyclin repeats are shown in the same colors. (B) Predicted three-dimensional structure of the N-terminal region of LmBrf1. Homology modeling was performed for LmBrf1 using the crystal structure from ScBrf1 as a template. A merge figure is also shown. All the structures are shown in the same colors presented in panel A. The quality of the models was estimated with Mod Eval server, showing a score of 0.70 (0.89 if we compare only the first and second cyclin repeats).
Figure 2
Figure 2
LmBrf1 is a nuclear protein. (A) Western blot analysis with cells that express the recombinant protein LmBrf1-PTP (Brf1-PTP (Prot C-TEV-Prot A)) and wild-type cells (WT). Membranes were incubated with an antibody against Prot C, which recognizes the PTP tag. As a loading control, a human α/β-tubulin antibody was used. (B) PTP-tagged LmBrf1 was detected by immunofluorescence with a rabbit anti-Prot C polyclonal antibody and an anti-rabbit immunoglobulin G (IgG) coupled with Alexa Fluor 568 (red). Nop56 was detected with a mouse anti-Nop56 antibody and an anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 (green). Nuclei (N) and kinetoplast (K) DNA were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 2 μm.
Figure 3
Figure 3
Half-lives of messenger RNAs (mRNAs) from Brf1, B double prime 1 (Bdp1), and RPC1. (A) Northern blots with total RNA from cells treated with actinomycin D (10 µg/mL) to inhibit transcription. RNA was isolated from logarithmic phase or stationary phase parasites at several time points (0, 1, 2, 4, 8, and 16 h) after addition of the drug. As a control, RNA from cells grown in the presence of dimethyl sulfoxide (10 µL/mL of culture), the solvent of actinomycin D, was included (lanes C). Hybridizations were performed with radioactive probes that corresponded to Brf1, Bdp1, RPC1, and α-tubulin. As loading control, membranes were re-hybridized with an 18S ribosomal RNA (rRNA) probe. (B) mRNA decay curves. Signals from the experiment shown in panel A and from two independent experiments were quantified and plotted. Results are presented from logarithmic-phase (blue line) and stationary-phase (red line) parasites. Error bars represent standard deviations.
Figure 4
Figure 4
Southern blot analysis of LmBrf1 single-knockout parasites. (A) Genomic DNA from a single-knockout clone (SKO) and wild-type parasites (WT) was digested with SalI and NotI. The probe corresponded to the 5′-targeting region of the LmBrf1 gene. The size of the observed bands is indicated. The larger fragments observed with DNA from the SKO clone digested with SalI correspond to partial digestion of the enzyme. (B) Restriction maps of the wild-type LmBrf1 locus (top map) and mutant loci with a copy of LmBrf1 replaced with the pac gene (bottom map). Sizes of predicted restriction fragments after digestion with SalI and NotI are shown. The location of the fragment employed as a probe is indicated with a gray bar.
Figure 5
Figure 5
Chromatin immunoprecipitation (ChIP) analysis of LmBrf1. (A) Schematic representation of the genes and promoter regions examined. Genes transcribed by Pol III, Pol II, and Pol I are shown in blue, yellow, and green, respectively. Maps are not to scale. (B) Chromatin from a clonal cell line that expresses the recombinant protein LmBrf1-PTP was precipitated with a ChIP-grade anti-Prot A antibody. Precipitated DNA was analyzed by qPCR. The results from three independent ChIP experiments, each analyzed by two qPCR reactions, are shown. Results are presented as percentage of input, corrected by subtracting corresponding values from negative control precipitations performed with a nonspecific antiserum. Error bars indicate standard errors.
Figure 6
Figure 6
Tandem affinity purification with L. major cells expressing LmBrf1-PTP. SDS-PAGE of proteins co-purified with LmBrf1-PTP. The asterisk denotes the recombinant protein. A control experiment with wild-type cells (WT) is also shown. The samples were analyzed in 4–15% Mini-PROTEAN Precast Protein Gels stained with SYPRO Ruby.
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