RESUMO
In the protist parasite Trypanosoma brucei, the small nuclear spliced leader (SL) RNA and the large rRNAs are key molecules for mRNA maturation and protein synthesis, respectively. The SL RNA gene (SLRNA) promoter recruits RNA polymerase II and consists of a bipartite upstream sequence element (USE) and an element close to the transcription initiation site. Here, we analyzed the distal part of the ribosomal (RRNA) promoter and identified two sequence blocks which, in reverse orientation, closely resemble the SLRNA USE by both sequence and spacing. A detailed mutational analysis revealed that the ribosomal (r)USE is essential for efficient RRNA transcription in vivo and that it functions in an orientation-dependent manner. Moreover, we showed that USE and rUSE are functionally interchangeable and that rUSE stably interacted with an essential factor of SLRNA transcription. Finally, we demonstrated that the T.brucei homolog of the recently characterized transcription factor p57 of the related organism Leptomonas seymouri specifically bound to USE and rUSE. Since p57 and its T.brucei counterpart are homologous to SNAP50, a component of the human small nuclear RNA gene activation protein complex (SNAPc), both SLRNA and RRNA transcription in T.brucei may depend on a SNAPc-like transcription factor.
Assuntos
Regiões Promotoras Genéticas/genética , RNA de Protozoário/genética , RNA Ribossômico/genética , RNA Líder para Processamento/genética , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Trypanosoma brucei brucei/genética , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Ligação Proteica , RNA Polimerase II/metabolismo , RNA de Protozoário/biossíntese , RNA Ribossômico/biossíntese , RNA Líder para Processamento/biossíntese , Fatores de Transcrição/química , Transcrição Gênica/genética , Ativação TranscricionalRESUMO
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.
Assuntos
Expressão Gênica , Glicoproteínas de Membrana/genética , RNA Polimerase I/metabolismo , Transcrição Gênica , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , DNA Ribossômico/genética , Regulação da Expressão Gênica , Genes de Protozoários , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
In the protist parasite Trypanosoma brucei, RNA polymerase (pol) I transcribes the large ribosomal RNA gene unit and, in addition, variant surface glycoprotein gene expression sites and procyclin gene transcription units. The multifunctional role of RNA pol I in this organism is unique among eukaryotes, but only its largest subunit TbRPA1 has been characterized thus far. We have recently established the procyclic cell line RPIC which exclusively expresses RNA pol I tagged with the protein C epitope at the TbRPA1 C-terminus. In the present study, we prepared RPIC cell extracts and immunopurified RNA pol I using anti-protein C affinity matrix under high stringency conditions. We were able to identify five specific polypeptides on a silver-stained polyacrylamide-SDS gel with apparent molecular weights of 200, 180, 55, 29, and 22 kDa. Interestingly, the second largest subunit, TbRPA2, is 42-58 kDa larger than counterparts of other organisms. We have cloned and sequenced the complete TbRPA2 cDNA and found an open reading frame for a polypeptide of 179.5 kDa. The deduced amino acid sequence of TbRPA2 contains a unique N-terminal domain of approximately 250 amino acids. By raising a polyclonal antibody against a N-terminal peptide sequence of TbRPA2, we could specifically detect this polypeptide in immunoblots showing that it co-purifies with epitope-tagged TbRPA1. Moreover, we identified the homologous gene sequence LmRPA2 in Leishmania major and found that it encodes a homologous extension domain. Therefore, the N-terminal extra domain in trypanosomatid RPA2 polypeptides may serve a parasite-specific function.
