Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei.
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-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing 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 trans splicing. However, efficient trans 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.
Siegel
T Nicolai
TN
Laboratory of Molecular Parasitology, Box 185, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Tan
Kevin S W
KS
Cross
George A M
GA
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000737:chemistry; D022821:RNA Splice Sites; D016053:RNA, Protozoan / Q000235:genetics*; D017421:Sequence Analysis; D020040:Trans-Splicing; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D011743:Pyrimidines; D022821:RNA Splice Sites; D016053:RNA, Protozoan; D008156:Luciferases; D001616:beta-Galactosidase