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J. Biol. Chem., Vol. 281, Issue 4, 1857-1867, January 27, 2006

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Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs^*

Ewa Grudzien, Marcin Kalek, Jacek Jemielity, Edward Darzynkiewicz, and Robert E. Rhoads1

From the Department of Biophysics, Warsaw University, Warsaw, 02-089, Poland and the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932

mRNA degradation predominantly proceeds through two alternative routes: the 5'3' pathway, which requires deadenylation followed by decapping and 5'3' hydrolysis; and the 3'5' pathway, which involves deadenylation followed by 3'5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp₃G, m⁷Gp₃G, or m₂^7,3'-O Gp₃G) and 3' termini (A₃₁,A₆₀, or G₁₆) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'3' versus 3'5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the - and -phosphate moieties, m₂^7,3'-OGpp_CH2pG and m₂^7,3'-OGp_CH2ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m₂^7,3'-OGpp_CH2pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m₂^7,3'-OGpp_CH2pG, but not m₂^7,3'-OGp_CH2ppG, were more stable in vivo, indicating that the 5'3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m₂^7,3'-OGpp_CH2pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.

Received for publication, August 18, 2005 , and in revised form, October 28, 2005.

^* This work was supported by Grant 1R03TW006446-01 from the National Institutes of Health (to R. E. R. and E. D.), Grant 2R01GM20818 from the National Institutes of Health (to R. E. R.), Grant 2 P04A 006 28 from the Polish Ministry of Science and Information (to E. D.), and Grant PBZ-059/T09/10 from the State 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad{at}lsuhsc.edu.

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