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

Mol Cell Biol. 2005 Nov;25(21):9586-94. doi: 10.1128/MCB.25.21.9586-9594.2005.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Genes, Reporter
  • Luciferases / genetics
  • Molecular Sequence Data
  • Pyrimidines / chemistry
  • RNA Splice Sites
  • RNA, Protozoan / genetics*
  • Sequence Analysis
  • Trans-Splicing*
  • Trypanosoma brucei brucei / genetics*
  • beta-Galactosidase / genetics


  • Pyrimidines
  • RNA Splice Sites
  • RNA, Protozoan
  • Luciferases
  • beta-Galactosidase