RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei

Abstract

In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to alpha-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, alphabeta-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

FIG. 1.

Physical maps of the RNA pol I largest-subunit locus in wild-type trypanosomes and the RPIC cell line. (Top) Structure of the RNA pol I largest-subunit (TbRPA1) locus in wild-type trypanosomes and plasmid pI-ble-KO for gene replacement. The large grey box represents the coding region of the TbRPA1 of T. brucei. a and b indicate the 5′ and 3′ ends, respectively, of TbRPA1-derived targeting sequences. The three white bars above the map indicate regions that were cloned into the pZJM construct for expression of dsRNA of TbRPA1. (Middle) Structure of the TbRPA1 locus in cell line pI-81 and plasmid pRPIC-hph used for gene integration. (Bottom) Structure of the TbRPA1 locus in the cell line RPIC. Abbreviations: Bl, phleomycin resistance gene; H, hph gene; T, αβ-tubulin intergenic region (53); H23, intergenic region of hsp70 gene 2 and 3 (14); BSK+, Bluescript SK(+) vector; prot C tag, protein C tag. Restriction enzymes: E, EcoRI; X, XbaI; S, SalI.

FIG. 2.

In vitro transcription analysis of RNA pol I-depleted extracts. (A) Immunodetection of TbRPA1. Nondepleted extracts prepared from wild-type (WT) and RPIC cells were separated on an SDS-5% polyacrylamide gel, blotted, and probed with the polyclonal anti-TbRPA1 antibody (left panel) or the monoclonal anti-protein C antibody (right panel). Arrows on the left indicate positions of protein marker bands. (B) RNA pol I depletion from extracts. Flowthrough fractions of extracts subjected to anti-protein C affinity chromatography in the absence (−Ca⁺⁺) or presence (+Ca⁺⁺) of calcium ions were analyzed with anti-TbRPA1 antiserum and, as a control, with anti-40K antiserum. In addition, a corresponding amount of eluate from the affinity matrix incubated with RPIC extract in the presence of calcium ions (IP) was loaded onto the gel. (C and D) In vitro transcription analysis of RNA pol I-depleted extract. Standard transcription reactions were carried out with wild-type (C) and RPIC (D) extracts treated with anti-protein C affinity matrix and template constructs GPEET-trm (GPEET), Rib-trm (rDNA), and VSG-trm (VSG). In each reaction, the control template SLins19 (SL) was cotranscribed. In a further control, vector DNA was transcribed in nondepleted cell extract. Transcription signals were obtained by primer extension analysis of RNA prepared from in vitro transcription reactions using³²P-end-labeled oligonucleotides GLESS_PE and SLtag. Extension products were separated on 6% polyacrylamide-50% urea gels and visualized by autoradiography. Arrows on the right point to correctly sized products. Marker, MspI-digested pBR322.

FIG. 3.

Effect of the TbRPA1 RNAi on cell growth. The growth curves of trypanosome cell lines PIds2.2, Pids1.5, and Pids0.8 were determined in the absence (open squares) or presence (solid squares) of 1 μg of tetracycline per ml (for the induction of dsRNA expression). Cells were continuously maintained at a log phase. If needed, medium can be added to expand the culture. The total number of cells in each culture was calculated at different time points.

FIG. 4.

Northern blot analysis of the effect of the TbRPA1 RNAi on mRNA levels. Approximately equal amounts (∼10 μg) of total RNA from cell lines 29-13 (lanes C), PIds2.2 (lanes 1), Pids1.5 (lanes 2), and Pids0.8 (lanes 3), cultured in the absence (−) or presence (+) of 1 μg of tetracycline per ml for 48 h, were separated in 1% formaldehyde-agarose gels. (Top panel) The blot was hybridized to a TbRPA1 coding region probe. The final posthybridizational wash was performed in 0.1 × SSC-0.1% SDS at 65°C. (Middle panel) Hybridization with the β-tubulin gene probe, indicating that similar amounts of RNA were loaded in each lane. (Bottom panel) Probes on the filter was stripped off and rehybridized with an RNA pol II largest-subunit probe.

FIG. 5.

Effect of the TbRPA1 RNAi on nascent RNA synthesis in procyclic form trypanosomes. (A) Nuclear run-on analysis. Nuclei were isolated from the PpIds1.5 cell line (left panel) and the 29-13 cell line (right panel) before (lanes −) and 60 h (lane +1) or 70 h (lane +2 and + on the right hand panel) after the addition of 1 μg of tetracycline per ml to induce dsRNA expression. Newly synthesized nascent RNA was labeled by the incorporation of [α-³²P]UTP and was hybridized with slot blots containing the following probes: T. brucei 18S rRNA gene (rDNA), the mini-exon clone CL103 (Mini-exon), the 5S rRNA probe (5S), the procyclin coding region clone CPT4 (Procyclin), the coding region of the αβ-tubulin gene (Tubulin), a 1.8-kb HindIII fragment from the coding region of the hsp70 gene (Hsp), and the VSG 118 ES promoter sequence (VSG-P). (B) Quantification of the relative transciptional efficiency of different genes. The radioactivity bound on the nitrocellulose filters from the left panel of panel A was quantified with a PhosphorImager. The transcriptional efficiency of each gene relative to that of the 5S RNA gene was determined for each nuclear preparation. The relative transcriptional efficiency of all genes in uninduced trypanosomes was arbitrarily set to 100.

FIG. 6.

Effect of the TbRPA1 RNAi on the TbRP1 protein level in procyclic form trypanosomes. Total protein lysates (∼5 × 10⁶ trypanosomes), derived from cell line 29-13 (lanes C), cell line PpIds0.8 (lanes 1), and cell line PpIds1.5 (lanes 2) incubated in the absence (lanes 0) or presence of tetracycline for 63 h (lanes 63), 72 h (lanes 72), and 78 h (lanes 78) were size separated in SDS-5% polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. The blots were probed with anti-TbRPA1 (top panel [TbRPA1]). The same blots were later probed with anti-Tb-29 (bottom panel [Tb-29] [26]) to demonstrate that the relative amounts of protein were loaded in each lane.