DIFFERENTIAL RESPONSE TO RNA TRANSSPLICING SIGNALS WITHIN THE PHOSPHOGLYCERATE KINASE GENE-CLUSTER IN TRYPANOSOMA-BRUCEI

In trypanosomatids, nuclear pre-mRNA splicing is exclusively a trans-s plicing reaction in which a capped, 39 nt exon, the mini-exon, is posi tioned 5' to an open reading frame. Differential RNA splicing might re flect specific mini-exon and 3' splice site interactions. To test this hypothesis, we compared the efficiency of mini-exon addition to three natural 3' splice acceptor sites (SASs) located within a single pre-m RNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive s equences on a polycistronic pre-mRNA. This pre-mRNA gives rise to uneq ual amounts of PGK A, B and C mRNAs. When the SAS from each gene was p laced upstream of the luciferase open reading frame and the resultant constructs transiently transfected into T. brucei procyclic cells, luc iferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels wer e indistinguishable when the B and C SASs were compared. After replaci ng luciferase with chloramphenicol acetyl transferase in the test cons tructs, enzyme activities were shown to directly correlate with mRNA a mounts. Thus, poor splicing efficiency accounts for the differential e xpression of the PGK A mRNA during PGK pre-mRNA maturation. This react ion appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.