Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing in Trypanosoma brucei

ABSTRACT

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 α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and transsplicing of the downstream α-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 α-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 α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-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 α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar tocis-splicing enhancers described in other systems.

ACKNOWLEDGMENTS

We thank the class of 1997 of the Biology of Parasitism Course in Woods Hole, Mass., for their enthusiasm, hard work, and critical comments and for initiating some of the experiments; Anna Polotsky and Helen Kwon for continuous and excellent technical support; and Susan Baserga, Joan Steitz, and Sandra Wolin for critical reading of the manuscript.

This work was supported by grant AI28798 from the National Institutes of Health to E.U. and by a Burroughs Wellcome Scholar Award in Molecular Parasitology to E.U. C.L.-E. was partially supported by the Consejo Nacional de Investigaciones Cientı́ficas y Tecnológicas (CONICIT) of Venezuela.