Trypanosoma brucei: cis-acting sequences involved in the developmental regulation of PARP expression

Abstract

The procyclic acidic repetitive protein (PARP or procyclin) of the parasitic protozoan Trypanosoma brucei is a developmentally regulated protein that shows extreme differences in its level of expression in different stages of the parasite's life cycle. Specifically, it is the major surface protein in the procyclic (insect) stage and, although the PARP gene is being actively transcribed in the mammalian bloodstream stage, there is no detectable PARP mRNA or protein in these cells. The 3'-untranslated region (UTR) of PARP, as well as other trypanosome genes, has the ability to confer the appropriate developmental regulation pattern onto chimeric reporter genes. To understand the mechanism of posttranscriptional regulation, selective replacement mutagenesis of the PARP mRNA 3'UTR was done to identify the cis-acting sequences involved in the down-regulation of this mRNA in bloodstream-form T. brucei. Transient transformation of constructs containing the PARP promoter and 5'UTR, the beta-glucuronidase coding region, and the selectively mutagenized or unaltered PARP 3'UTR were performed in procyclic and bloodstream T. brucei. The results of the reporter gene assays on the transformed cells indicate that there are at least two elements in the PARP 3'UTR which in bloodstream cells are involved in regulation of PARP expression and which appear to function as negative elements. In procyclic cells, there are two regions in which mutagenesis indicates positive cis-regulatory sequences, one of which has been previously defined (A. Hehl et al., 1994, Proc. Natl. Acad. Sci. USA 91, 370-374). These results indicate that multiple cis-acting elements within the PARP 3'UTR are involved in the developmental regulation of PARP expression and that regulation is controlled in a complex manner, presumably involving several cellular trans-acting factors.