RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei.

Abstract

We show that the ribosomal RNA (rRNA) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan Trypanosoma brucei. The rRNA promoter was characterized by: (i) point mutations at the rRNA transcription initiation site which completely abolished its promoter function in transient CAT transformation assays; (ii) the alpha-amanitin resistance of transcription of rRNA promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) the nucleolar location of neo RNA, synthesized under the control of the rRNA promoter. The rRNA promoter-derived CAT mRNA required a 3' splice acceptor site and the neo mRNA was trans-spliced and polyadenylated. In situ hybridization revealed neo RNA at the nucleolus in stably transformed trypanosomes in which rRNA promoter-neo constructs were integrated either at a rRNA locus or at a locus for the procyclic acidic repetitive protein (PARP) coding genes. We postulate that trans-splicing, by uncoupling the requirement for transcription of protein-coding genes by RNA polymerase II, allows RNA polymerase I mediated protein-coding gene transcription, presumably because a 5' cap can be transferred to the pre-mRNA by trans-splicing.