TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

Abstract

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

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

“…Immunofluorescence assays showed that LmBrf1 is a nuclear protein ( Figure 2 ). A similar punctate nuclear signal was reported for other Pol III transcription factors in L. major and T. brucei , including Bdp1 [ 33 ] and Maf1 [ 41 ], as well as for the L. major 5S rRNA genes [ 29 ]. Even though the vast majority of LmBrf1 signal is nucleoplasmic, a small part of the immunostaining colocalized with Nop56 in the nucleolar periphery, which could support the association of LmBrf1 with the rRNA transcription unit that was found in ChIP experiments ( Figure 5 ) and the possible interaction with proteins involved in Pol I transcription ( Table 1 ), as discussed below.…”

“…As shown in Figure 2 B, LmBrf1 is localized in the nucleus of the parasites, as anticipated for a transcription factor. The nuclear distribution of LmBrf1 is very similar to that of LmBdp1 [ 33 ]. Notably, a small fraction of LmBrf1 seems to colocalize with the nucleolar protein Nop56 in the periphery of the nucleolus (in yellow in the Merge panels of Figure 2 B).…”

“…In other organisms, including yeast, Brf1 is an essential protein [ 52 ]. Similarly, LmBdp1 is an essential molecule for the viability of L. major promastigotes [ 33 ]. Other strategies, such as CRISPR/Cas9 genome editing, should be performed to demonstrate the essentiality of LmBrf1 for L. major growth.…”

“…L. major promastigotes from strain MHOM/IL/81/Friedlin (LSB-132.1) were grown in BM medium (1× M199 medium pH 7.2 containing 10% heat-inactivated fetal bovine serum, 9.5 g/L brain heart infusion, 40 mM HEPES, 0.01 mg/mL hemin, 0.0002% biotin, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 1× l -glutamine) at 26 °C and harvested in the mid-logarithmic or stationary phase. Transfection of promastigotes with the episomal LmBrf1-PTP vector was performed by electroporation as previously described [ 33 ]. Briefly, 4 × 10 7 promastigotes in 0.4 mL of electroporation buffer (25 mM HEPES, 120 mM KCl, 0.15 mM CaCl 2 , 5 mM MgCl 2 , 10 mM KH 2 PO 4 , 10 mM K 2 HPO 4 , and 2 mM EDTA, pH 7.6) were transfected with 10 μg of circular plasmid DNA by electroporation at 1600 V, 50 μF, and 25 Ω (BTX Electro Square Porator ECM 830).…”

“…The SNAP50 subunit of the SNAP complex was also found to associate with tRNA and snRNA genes [ 32 ]. A recent report indicates that TFIIIB subunit Bdp1 is an essential protein required for Pol III transcription of tRNAs, snRNAs, and 5S rRNA in L. major [ 33 ]. In T. brucei , tandem affinity purification experiments with a Prot C-TEV-Prot A (PTP)-tagged version of TBP demonstrated that Brf1 co-purified with TBP [ 34 ].…”