Characterization of subunits of the RNA polymerase I complex in Trypanosoma brucei

The Trypanosoma brucei homologue of the RNA polymerase I (RNA Pol I) subunit Rpa12p of Saccharomyces cerevisiae was cloned and characterized. This protein did not appear to be essential for growth in either bloodstream or procyclic forms of the parasite. Trypanosomes expressing a C-terminal tagged version of TbRPA12 were generated in order to purify RNA Pol I from both developmental stages. Tandem affinity purification (TAP) revealed a number of proteins associating with TbRPA12, some of which appeared to be stage-specific. Mass spectrometry allowed the identification of four subunits in addition to TbRPA12, namely TbRPA1, TbRPA2, TbRPC40 and one isoform of TbRPB5 (Tb1RPB5), as well as an unknown 30kDa protein and histones H2A and H3. Whereas these studies demonstrated that TbRPA1 was phosphorylated, no evidence for phosphorylation of TbRPA2 was found.     

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.     

Transcription by T7 RNA polymerase of DNA containing abasic sites

The effects of abasic (AP) sites on RNA synthesis were studied in vitro, using T7 RNA polymerase and a plasmid template containing a T7 promoter. The presence of increasing numbers of AP sites caused a progressive decline in RNA synthesis. The average RNA chain length, calculated from the ratio of initiation to chain elongation, decreased with increasing numbers of AP sites, revealing that complete blocks must occur during synthesis. The probability that RNA polymerase would be blocked at an AP site in the DNA template strand was estimated to be 0.3 in our experimental conditions. These results demonstrate that RNA synthesis by T7 RNA polymerase is inhibited by AP sites and that readthrough of the lesion occurs more frequently than premature chain termination. Chemical reduction of AP sites in the template did not change the block/bypass pattern. © 1994 Wiley-Liss, Inc.     

The 7SL RNA homologue of Trypanosoma brucei is closely related to mammalian 7SL RNA.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by a signal recognition particle, a small ribonucleoprotein (RNP) containing 7SL RNA. We have cloned and sequenced the gene coding for the Trypanosoma brucei 7SL RNA homologue and found that its sequence shows the highest degree of similarity to the human 7SL RNA sequence. In keeping with the prototype secondary structure of eukaryotic 7SL RNA, the trypanosome 7SL RNA secondary structure can be folded into four domains. The 7SL RNP, which sediments at approximately 11S on sucrose density gradients, was partially purified using column chromatography. A particle containing a 76-nucleotide-long RNA co-purified with the 7SL RNP; however, these particles did not co-fractionate by non-denaturing polyacrylamide gel electrophoresis.     

Messenger RNA processing sites in Trypanosoma brucei

In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .     

Control of experimental Trypanosoma brucei infections occurs independently of lymphotoxin-alpha induction

Trypanosome infections are marked by severe pathological features, including anemia, splenomegaly, and suppression of T-cell proliferation. We have used lymphotoxin-alpha-deficient (LT-alpha(-/-)) mice, as well as LT-alpha-tumor necrosis factor-double-deficient (LT-alpha(-/-) TNF(-/-)) mice, to analyze the contributions of these related cytokines in both induction of trypanosomosis-associated immunopathology and infection control. Moreover, as the cytokine-deficient mice used have no detectable lymph nodes and lack germinal-center formation upon immune stimulation, we have analyzed the functional importance of both the lymph nodes and spleen during experimental Trypanosoma brucei infections. First, we show that the absence of LT-alpha does not significantly alter early trypanosomosis development or pathology but does result in better control of late-stage parasitemia levels and slightly prolonged survival. This increased survival of infected LT-alpha(-/-) mice coincides with the appearance of increased chronic-stage anti-trypanosome immunoglobulin M (IgM)-IgG2a serum titers that are generated in the absence of functional peripheral lymphoid tissue and do not require germinal-center formation. Second, we show that splenectomized mice control their parasitemia to the same extent as fully immune-competent littermates. Finally, using LT-alpha(-/-) TNF(-/-) double-deficient mice, we show that in these mice T. brucei infections are very well controlled during the chronic infection stage and that infection-induced pathology is minimized. Together, these findings indicate that while increased IgM-IgG2a anti-trypanosome antibody titers (generated in the absence of LT-alpha, peripheral lymph nodes, and germinal-center formation) coincide with improved parasitemia control, it is TNF that has a major impact on trypanosomosis-associated immunopathology.