Exonic sequences in the 5' untranslated region of alpha-tubulin mRNA modulate trans splicing in Trypanosoma brucei

Previous studies have identified a conserved AG dinucleotide at the 3' splice site (3'SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei alpha-tubulin 3'SS region is required to specify accurate 3'-end formation of the upstream beta-tubulin gene and trans splicing of the downstream alpha-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3'SS identification. Our results indicate that a minimal alpha-tubulin 3'SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the alpha-tubulin 3'SS is dependent upon the presence of exon sequences. Furthermore, beta-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace alpha-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the alpha-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.