mRNA degradation predominantly proceeds through two alternative routes: the 533 pathway, which requires deadenylation followed by decapping and 533 hydrolysis; and the 335 pathway, which involves deadenylation followed by 335 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. The 5Ј terminus of all eukaryotic cellular mRNAs is modified with a 5Ј-5Ј m 7 GTP-containing cap (1). Caps fulfill a variety of functions in the synthesis, translation, and degradation of mRNA. The presence of the 5Ј cap structure increases both the accuracy and efficiency of pre-mRNA splicing (2, 3). The cap on pre-mRNA interacts with the nuclear cap-binding complex, which remains bound and plays an active role during RNA processing and export (4). In the cytosol, the cap structure is required for efficient translation of mRNA. The cap is specifically recognized by the translational initiation factor eIF4E (5, 6). Binding of eIF4E to the cap occurs during formation of the 48 S initiation complex, which is rate limiting for translation initiation under normal conditions (7,8). Finally, the cap serves as one determinant of mRNA degradation. Capped mRNAs are more stable than their uncapped counterparts (9). The cap structure helps to protect RNA from degradation by 5Ј33Ј-exonucleases located in the cytosol and nucleus, as demonstrated in both Saccharomyces cerevisiae (10) and mammalian cells (11,12).A second stability element in mRNA is the 3Ј-terminal poly(A) tract. PABP 2,3 binds to poly(A) and is essential for the stability provided by this element, protecting mRNA against exonucleolytic degradation (12-15). PABP also binds to the N terminus of eIF4G (16) and stabilizes the eIF4G⅐eIF4E complex, enhancing translational reinitiation (17, 18). The stimulation conferred by the cap and poly(A) tract are synergistic rather than additive (19,20). Thus, for both translation and degradation of mRNA, elements binding to the 5Ј and 3Ј termini act cooperatively and in close proximity.There are two major pathways by which polyadenylated mRNA is degraded in eukaryotic cells, a 5Ј33Ј pathway and a 3Ј35Ј pathway, as well as two specialized routes for aberrant mRNA degradation (21). In both the 5Ј33Ј and 3Ј35Јpathways, shortening of the poly(A) tract initiates mRNA decay. There are several mechanism of deadenylation (21), but one of them involves a poly(A)-specific ribonuclease (22), an enzyme tha...

Gp 3 G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.

The eukaryotic initiation factor eIF4E binds the mRNA 5 0 cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m 7 GTP and m 7 GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.

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