A divergent transcription factor TFIIB in trypanosomes is required for RNA polymerase II-dependent spliced leader RNA transcription and cell viability

Abstract

Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

FIG. 1.

Multiple sequence alignment of trypanosome TFIIB and homologs in other eukaryotes that contain two repeat regions. (A) The amino-terminal sequence alignment shows the similarity of Trypanosoma brucei TFIIB (Tb, XM_821873.1) with the homologs from Trypanosoma cruzi (Tc, AAHK01001127.1), Homo sapiens (Hs, Q00403), Saccharomyces cerevisiae (Sc, AAB68135), the archeael Pyrococcus furiosus (Pf, AE010241.1), and Oryza sativa (Os, AC105731.2). Black shading signifies complete conservation, gray shading with white lettering signifies greater than 80% conservation in that column, and gray shading with black letters signifies greater than 60% conservation in that column. The zinc ribbon region is designated by a bar. Residues known to be important for transcription start site selection are indicated by vertical arrows. (B) Alignment of the T. brucei TFIIB region corresponding to the first repeat of human TFIIB.

FIG. 2.

TbTFIIB interacts with RNA polymerase II and TbTBP. (A) GST and GST-TbTFIIB pulldown assays. Those proteins captured by GST or GST-TbTFIIB were probed with antibodies specific for RNA polymerase II (largest subunit), TbTBP, TbSNAP50, TbPBP-2, or TbPARN, as identified by arrows. For Western blot analysis, the supernatant contains one-seventh the volume of that loaded for the captured proteins. (B) Coimmunoprecipitation of proteins interacting with TbTFIIB. Shown are Western blot analysis results for the RNA polymerase II largest subunit, TbPARN, and TbPBP-2. (C) A high-salt titration of the TbTFIIB-TbTBP interaction, from 0.7 to 1.0 M KCl. Western blot analysis of GST alone or the GST-TbTFIIB pulldown assay using recombinant His₆-TbTBP is shown. TbTBP is indicated by an arrow.

FIG. 3.

TbTFIIB is required for SL RNA gene transcription. (A) Schematics of the wild-type SL RNA and U6 snRNA gene promoters and portions of the transcribed tagged SL RNA and U6 snRNA sequences are shown. (B) Western blot analysis of preimmune-depleted and TbTFIIB antibody-depleted nuclear extracts. Essentially, all TbTFIIB is depleted after a single incubation of antibody with extract. This immunodepletion does not remove all TbRNA Pol II, TbTBP, or TbSNAP50 from the extract. (C) In vitro transcription reactions. Transcription activity was lost in the TbTFIIB-depleted extract (compare lane 3 with lane 1 or 2). Restoration of activity was observed with the addition of 200 or 400 ng of recombinant TbTFIIB (lanes 4 and 5). The addition of 400 ng of BSA served as a negative control (lane 6).

FIG. 4.

TbTFIIB is essential for cell viability. (A) Growth curves for clonal cell lines under noninduced (open circles) and tetracycline-induced (closed circles) conditions. Parasite number was determined daily and is represented in a logarithmic scale. (B) Western blot analysis was used to follow the depletion of TbTFIIB. Proteins, separated on SDS-10% polyacrylamide gels, were detected with TbTFIIB antibodies (indicated by arrows). Each lane contains protein from 6 × 10⁶ parasites. A protein unrelated to TbTFIIB (indicated by an asterisk) and recognized by the antiserum in a nonspecific way served as a loading control. (C) Western blot analysis of nuclear extract prepared from cells on day 4 reveals that while TbTFIIB is knocked down, TbRNA Pol II, TbTBP, and TbSNAP50 all remain. (D) In vitro transcription studies performed with the nuclear extract from day 4 show that transcription of SL RNA was unhindered in the noninduced extract but was abolished with the tetracycline-induced knockdown of TbTFIIB (compare lanes 1 and 2). The addition of either 200 or 400 ng recombinant TbTFIIB (lanes 3 and 4) to induced extract but not BSA (lane 5) restored transcriptional activity.