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J. Biol. Chem., Vol. 279, Issue 44, 45573-45585, October 29, 2004

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Contribution of Trans-splicing, 5' -Leader Length, Cap-Poly(A) Synergism, and Initiation Factors to Nematode Translation in an Ascaris suum Embryo Cell-free System^*

Sabbi Lall, Cassandra C. Friedman, Marzena Jankowska-Anyszka¶, Janusz Stepinski||, Edward Darzynkiewicz||, and Richard E. Davis**

From the Department of Biology, City University of New York Graduate Center, College of Staten Island, Staten Island, New York 10314 and the ^¶Department of Chemistry and ^||Department of Biophysics, University of Warsaw, 02-093 Warsaw, Poland

Trans-splicing introduces a common 5' 22-nucleotide sequence with an N-2,2,7-trimethylguanosine cap (m ^2,2,7₃GpppG or TMG-cap) to more than 70% of transcripts in the nematodes Caenorhabditis elegans and Ascaris suum. Using an Ascaris embryo cell-free translation system, we found that the TMG-cap and spliced leader sequence synergistically collaborate to promote efficient translation, whereas addition of either a TMG-cap or spliced leader sequence alone decreased reporter activity. We cloned an A. suum embryo eIF4E homolog and demonstrate that this recombinant protein can bind m⁷G- and TMG-capped mRNAs in cross-linking assays and that binding is enhanced by eIF4G. Both the cap structure and the spliced leader (SL) sequence affect levels of A. suum eIF4E cross-linking to mRNA. Furthermore, the differential binding of eIF4E to a TMG-cap and to trans-spliced and non-trans-spliced RNAs is commensurate with the translational activity of reporter RNAs observed in the cell-free extract. Together, these binding data and translation assays with competitor cap analogs suggest that A. suum eIF4E-3 activity may be sufficient to mediate translation of both trans-spliced and non-trans-spliced mRNAs. Bioinformatic analyses demonstrate the SL sequence tends to trans-splice close to the start codon in a diversity of nematodes. This evolutionary conservation is functionally reflected in the optimal SL to AUG distance for reporter mRNA translation in the cell-free system. Therefore, trans-splicing of the SL1 leader sequence may serve at least two functions in nematodes, generation of an optimal 5'-untranslated region length and a specific sequence context (SL1) for optimal translation of trimethylguanosine capped transcripts.

Received for publication, July 6, 2004 , and in revised form, August 18, 2004.

^* This work was supported by National Institutes of Health Grants AI49558, AI043300, and AI039714 and City University of New York-College of Staten Island startup funds (to R. E. D.) and by Grants PBZ-KBN-059/T09/10 and KBN 3 P04A 021 25 from the Polish Committee for Scientific Research to (E. D.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Tables 1–3.

This article was selected as a Paper of the Week.

Current address: Dept. of Biology, New York University, New York, NY 10012.

^** To whom correspondence should be addressed. Current address: UCHSC at Fitzsimons, Depts. of Biochemistry and Molecular Genetics and Pediatrics, Mail Stop 8101, P. O. Box 6511, 12801 East 17th Ave., Aurora, CO 80045. Tel.: 303-724-3226; Fax: 303-724-3215; E-mail: richard.davis{at}uchsc.edu.

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