Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

Abstract

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...

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

“…This assumption is based on the finding that protection of the 5 0 end of an RNA against cleavage by Dcp2 shifts the balance of 5 0 -3 0 and 3 0 -5 0 degradation to the latter. 25 In a recent report that is consistent with our data, it was shown that ectopic CD40L expression in immature DCs is higher from post-transcriptionally capped RNA compared with ARCA-capped RNA. 26 However, b-S-ARCA(D1) (or D2) was not tested by these investigators, and thus the superiority of b-S-ARCA(D1)-capped RNAs over post-transcriptionally capped RNA on protein expression and RNA stability in immature DCs remained unrevealed.…”

“…The stabilization of b-S-ARCA(D1)-capped RNA would thus be explained by efficient binding of eIF4E to the 5 0 end, as previously shown. 25 In contrast, in mature DCs the influence of RNA stabilization and affinity for eIF4E seems to be the opposite, because b-S-ARCA(D2)-capped RNA gives highest protein synthesis. In addition, our data can be observed as an indication for domination of 5 0 -3 0 rather than 3 0 -5 0 degradation routes in immature DCs.…”

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

“…Stabilities were predicted using Mfold version 3.0 (60). RNA was synthesized by transcribing plasmids with T7 RNA polymerase (61). Capped mRNAs were produced by lowering the GTP concentration from 0.5 to 0.1 mM and including the ARCA at 1 mM (62), which prevents the cap from being incorporated in the reverse orientation (51).…”

Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism

“…Synthesis of the SB reporter RNA lacking a 5Ј cap structure was completed by using a Ribomax (Qiagen) RNA probe kit. Reporter RNAs synthesized with host decapping enzyme cleavage-resistant cap analog were transcribed as described previously (19). For radiolabeling of in vitro-transcribed RNA, transcripts were synthesized as described above in the presence of [ 32 P] ATP (Amersham), precipitated as described above, and used directly for electroporation.…”

“…This result suggested that an IFN-␣/␤-induced decapping activity might account for the translation inhibition observed. To test this, we electroporated IFN-␣/␤-treated and untreated cells with transcripts produced using a cap structure (m 27,3Ј-O Gpp CH2 pG) that has been shown to be highly resistant to the Dcp2 decapping enzyme in vitro and to significantly retard RNA degradation in cells (19). The effects of IFN-␣/␤ priming on translation were similar in magnitude if the cleavage-resistant cap was used for transcription (Fig.…”