Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes

Abstract

Messenger RNAs of higher eukaryotes share a functionally essential 5' monomethyl CAP structure generated during a reaction that is linked exclusively to RNA polymerase II transcription. In unicellular parasites belonging to the Kinetoplastida, however, mRNAs acquire their 5' CAP through a trans-splicing reaction which effectively uncouples pol II transcription and capping. Consequently functional mRNAs can be produced by endogenous RNA polymerase I. Here we demonstrate the extension of this flexibility to heterologous bacteriophage polymerases. Transgenic Trypanosoma brucei cell lines stably expressing functional, nuclearly localized T3 or T7 RNA polymerase were established and assayed using reporter plasmids bearing the corresponding phage promoters. In these cell lines the levels of phage promoter-driven gene expression ranges from one half to greater than 5 times that mediated by endogenous pol I. Analysis of 5' ends of transcripts synthesized by the T7 polymerase revealed that they are trans-spliced. Thus the usual eukaryotic link between mRNA production and pol II transcription can be by-passed by the introduced phage polymerases, thereby significantly expanding the critically small panel of promoters currently available for exploitation in reverse genetic approaches in T. brucei.