Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei

Abstract

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5′ splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts],3′ splice sites (3′SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3′SS, we constructed a luciferase-β-galactosidase double-reporter system. By testing ∼90 sequences, we demonstrated that the optimum poly(Y) tract length is ∼25 nucleotides. Interspersing a purely undine-containing poly(Y) tract with cytidine resulted in increased ironssplicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3′SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in irons splicing. However, efficient irons splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3′SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome. Copyright © 2005, American Society for Microbiology. All Rights Reserved.