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Molecular and Cellular Biology, November 2005, p. 9586-9594, Vol. 25, No. 21
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.21.9586-9594.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

T. Nicolai Siegel, Kevin S. W. Tan, and George A. M. Cross^*

Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York

Received 28 April 2005/ Returned for modification 6 July 2005/ Accepted 9 August 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-ß-galactosidase double-reporter system. By testing 90 sequences, we demonstrated that the optimum poly(Y) tract length is 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.

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* Corresponding author. Mailing address: Laboratory of Molecular Parasitology, Box 185, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Phone: (212) 327-7571. Fax: (212) 327-7845. E-mail: george.cross{at}rockefeller.edu.

Present address: Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.

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Molecular and Cellular Biology, November 2005, p. 9586-9594, Vol. 25, No. 21
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.21.9586-9594.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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