African trypanosomes are unicellular eukaryotic flagellates which cause serious diseases in human and livestock (sleeping sickness, Nagana). They have an intricate life cycle involving transmission between a mammalian host and an insect vector, the tsetse fly. All mRNAs in trypanosomatids are generated by the joining of two separate RNA precursor transcripts via trans-splicing. As a result, the 5' ends of all mRNAs in T. brucei consist of a common, capped 39-nt non-coding mini-exon. Importantly, trans-splicing allows the 5' cap to be added to each mRNA post-transcriptionally, and thus may have uncoupled the production of the capped mRNA by RNA polymerase (pol) II. Comparing the alpha-amanitin sensitivity of trypanosome protein-coding gene transcription revealed that the Variant cell Surface Glycoprotein (VSG) gene expression site (ES) and Procyclic Acidic Repetitive Protein (PARP or procyclin) genes are transcribed by an RNA pol that is resistant to alpha-amanitin, a characteristic of transcription by RNA pol I. A large body of indirect evidence, now available, supports the notion that RNA pol I, which normally only transcribes rRNA genes, transcribes the VSG and PARP genes. In this proposal, we will perform a thorough and conclusive analysis of the RNA polymerase that transcribes the VSG and PARP genes and study mechanisms of differential transcription of these two genes. First, we will characterize the 5' end structure of primary transcripts. This study will reveal whether RNA pol II primary transcripts are capped and whether an RNA pol associated capping activity is present in trypanosomes. The finding of a capped 5' end or tri-or di-phosphate at the 5' end of the primary transcripts can unambiguously settle whether RNA pol I or pol II controls the transcription of VSG and PARP genes. Second, trypanosome pol I point mutations that result in temperature sensitive mutants and trypanosome pol I knock-out cell lines will be established. These cell lines will be used to further address the nature of the VSG ES and the PARP gene transcription. Third, we will study mechanisms of differential transcription of surface coat protein genes in T. brucei.
Aims I and II will resolve the question whether transcription of the surface coat protein genes of T. brucei is mediated by RNA pol I. The understanding of the RNA pol controlling the transcription of the VSG and PARP genes is crucial for the study of the differential transcription of the VSG, PARP and rRNA genes during the parasite development and when integrated at different chromosomal contexts.
