Requirement of a polypyrimidine tract for trans-splicing in trypanosomes: discriminating the PARP promoter from the immediately adjacent 3' splice acceptor site

Abstract

We studied sequence requirements for trans-splicing at the 3' splice acceptor site of a procyclic acidic repetitive protein (PARP) coding gene in trypanosomes. In transient CAT transfection assays with linker scanning (LS) mutants in a PARP promoter--3' splice acceptor site--CAT construct, minor differences in the sequence composition of the polypyrimidine tract (nt -36 to -5 with respect to the 3' splice acceptor site) severely affected the CAT activity. Analysis of steady-state CAT RNA in stably transformed trypanosomes revealed that the LS mutations had indeed affected the pre-mRNA splicing efficiency. The data indicate that mini-exon addition is not required simply for maturation of polycistronic pre-mRNA but is also essential for the generation of functional mRNA from monocistronic genes, since unspliced monocistronic pre-mRNA did not accumulate or allow synthesis of CAT. We postulate that mini-exon addition at polycistronically transcribed genes, which can have drastically different polypyrimidine tracts at each of their 3' splice acceptor sites, can occur with different efficiencies for each gene of the array thus affecting mRNA abundance.