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J. Biol. Chem., Vol. 278, Issue 40, 38269-38275, October 3, 2003

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The Leishmania tarentolae Spliced Leader Contains Determinants for Association with Polysomes^*

Gusti M. Zeiner , Nancy R. Sturm and David A. Campbell 

From the Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90095-1489

In kinetoplastids, every nuclear-derived mRNA contains an identical 39-nucleotide (nt) spliced leader at its 5'-terminus. The spliced leader is derived from substrate spliced leader RNA and joined to pre-mRNA by trans-splicing, thus providing mature mRNAs with an m⁷G cap and additional methylations referred to as cap 4. It was shown previously that mutations spanning nucleotides 10–39 of the spliced leader did not affect substrate spliced leader RNA transcription or trans-splicing in Leishmania tarentolae (Saito, R. M., Elgort, M. G., and Campbell, D. A. (1994) EMBO J. 13, 5460–5469). In this study we examined these sequences for a possible role in translation by assaying the association of mRNAs, which possess mutated spliced leaders, with polysomes. For the nt 28–39 mutated spliced leaders, both the substrate spliced leader RNA and the spliced leader demonstrated a wild-type methylation pattern; spliced nt 28–39 mRNA was found in polysomes. Thus, the nt 28–39 region conserved primary sequence is not a determinant of polysome association. An undermethylated cap 4 structure was present on substrate and mRNA spliced leaders in nt 20–29 mutated exons; nt 20–29 mRNA was not present in polysomes. A differential pattern of cap 4 methylation was seen between the nt 10–19 substrate spliced leader RNA and the nt 10–19 spliced leaders found in the poly(A)⁺ population of RNA; the nt 10–19 mRNA was not seen in polysomes. Undermethylated spliced leaders did not associate efficiently with polysomes, suggesting a requirement for the cap 4 and/or primary sequence of the spliced leader in translation. This is the first report demonstrating that the spliced leader contains critical structural or sequence determinants for association with polysomes and, hence, translation.

Received for publication, April 24, 2003 , and in revised form, July 17, 2003.

^* This work was supported by National Institutes of Health Grant AI34536 (to D. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Predoctoral candidate and recipient of Microbial Pathogenesis Training Grant 2-T32-AI-07323.

To whom correspondence should be addressed: Dept. of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, 609 Charles E. Young Dr. E., Los Angeles, CA 90095-1489. Tel.: 310-206-5556; Fax: 310-206-5231; E-mail: dc{at}ucla.edu.

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