Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Abstract

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 (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) 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 alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, 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 m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.