Differential response to RNAtrans-splicing signals within the phosphoglycerate kinase gene cluster inTrypanosoma brucei

In trypanosomatids, nuclear pre-mRNA splicing is exclusively a trans-splicing reaction in which a capped, 39 nt exon, the mini-exon, is positioned 5' to an open reading frame. Differential RNA splicing might reflect 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-mRNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive sequences on a polycistronic pre-mRNA. This pre-mRNA gives rise to unequal amounts of PGK A, B and C mRNAs. When the SAS from each gene was placed upstream of the luciferase open reading frame and the resultant constructs transiently transfected into T. brucei procyclic cells, luciferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels were indistinguishable when the B and C SASs were compared. After replacing luciferase with chloramphenicol acetyl transferase in the test constructs, enzyme activities were shown to directly correlate with mRNA amounts. Thus, poor splicing efficiency accounts for the differential expression of the PGK A mRNA during PGK pre-mRNA maturation. This reaction appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.