REVIEWS
CAP ANALOGUES
Dinucleotides such as 7-meGpppG or GpppG that resemble the 5′ cap structure of messenger RNAs. They can be used to analyse the specificity of cap-binding proteins or to compete them away from the 5′ cap structure.
Poly(A) removal triggers rapid cleavage of the 5′ cap by a decapping enzyme (Dcp1)11,12. Biochemical and genetic analyses have identified several factors involved in decapping, including Dcp2 (REF. 13), Vps16 (REF. 14), Pat1 (REF. 15,16) and the Lsm proteins17,18 (TABLE 1). Although mutations in each of the genes encoding these factors prevent decapping, their roles in either catalysing or regulating this reaction remain to be determined. After decapping, the body of the transcript is degraded by the 5′ exonuclease Xrn1 (REF. 19). There are 3′ exonuclease activities in yeast, but they seem to have only minor functions in normal mRNA decay20. Deadenylation-dependent mRNA decay is important for regulating transcript stability in mammalian cells21. A poly(A)-specific deadenylating nuclease — initially termed DAN, but subsequently designated as PARN (poly(A) ribonuclease) — has been purified and biochemically characterized from both mammalian cells and Xenopus laevis oocytes22–24. The subsequent steps of mRNA decay in multicellular organisms are not
Decapping enzyme 4E
RN PA
4G
PABP
AAAAAAAAAAAAAAAAAAAA
Deadenylation
AA
AA
AA
AA
Decapping
Figure 2 | The deadenylase as an inhibitor of translation initiation and decapping. During translation, the mRNA is thought to be circularized by its interaction with the translationinitiation factors eIF4E (4E), eIF4G (4G) and the poly(A)-binding protein (PABP). The eIF4E protein binds to the 5′ cap structure and this interaction is promoted by its binding to eIF4G. eIF4G is a large protein that can interact with both eIF4E and PABP simultaneously. This conformation protects the 5′ and 3′ ends of the mRNA from attack by the deadenylase and decapping enzymes. We postulate that the deadenylase can somehow invade this closed loop and interact with the cap while simultaneously removing the poly(A) tail. The interaction of poly(A) ribonuclease (PARN) with the cap perpetuates the closed loop and thereby blocks both translation initiation and decapping. When poly(A) shortening is complete, PARN dissociates, allowing the decapping enzyme to hydrolyse the 5′ cap of the message.
as well defined as in yeast, owing to the transient nature of intermediate decay products. However, decapped mRNA decay intermediates have been isolated from mouse liver cells25. Moreover, a decapping activity that can be inhibited by the poly(A) tail has recently been characterized in HeLa cell extracts26, indicating that decapping might be an important step in the deadenylation-dependent turnover of mammalian mRNAs. Although mammalian homologues of the yeast Lsm proteins27 and the Dcp2 protein13 have been identified, their functions remain to be determined. It is also not clear whether the body of deadenylated and decapped RNA is degraded by 5′ or 3′ exonucleases. Although a mammalian homologue of yeast Xrn1 exoribonuclease has been identified28, only 3′ exonuclease activities have been observed in vitro29–31. The poly(A) tail inhibits mRNA decay through its interaction with the poly(A)-binding protein (PABP)32 (FIG. 2). As well as binding poly(A), PABP interacts with a specific region of the translation-initiation factor eIF4G, which in turn forms a ternary complex with the cap-binding protein eIF4E33 (FIG. 2). This complex circularizes the mRNA in vitro34, can promote translation, and might simultaneously stabilize mRNAs by preventing access of deadenylating and decapping enzymes to their targets. Indeed, evidence from recent analyses indicates that accessibility of the 5′ cap to both deadenylating and decapping activities might be a major determinant of mRNA stability26,35,36. But the role of PABP in deadenylation remains to be established. PABP inhibits deadenylation in mammalian cell-free assays32,37 and when overexpressed in Xenopus oocytes39, consistent with its function as a negative regulator of deadenylation. By contrast, yeast strains lacking the PAB1 gene actually show slower deadenylation, suggesting that Pab1 might contribute to optimal poly(A) shortening38. Biochemical studies have shown that the deadenylating nuclease PARN binds directly to the 5′ cap structure on a substrate RNA, and that this interaction stimulates deadenylase activity both in vitro and in vivo36. In addition, both CAP ANALOGUES and eIF4E inhibit PARN in vitro by competing for binding to the 5′ cap35,36. Intriguingly, cap analogues stimulate decapping activity by preventing the binding of both PARN and eIF4E26,35. So one can imagine a sequential process in which an initial event must destabilize the poly(A)–PABP and cap–eIF4E complexes, allowing PARN to bind simultaneously to both the 5′ cap and the 3′ poly(A) tail. We suggest that the interaction of PARN with the cap and poly(A) tail can inhibit decapping, even if the ternary eIF4E, eIF4G and PABP complex is disrupted. Once deadenylation is complete, PARN dissociates and the decapping enzyme can now recognize the cap. These observations indicate that the translational efficiency of an mRNA might be directly coupled to mRNA decay, and that both processes are determined by interactions assembled on the 5′ cap and 3′ poly(A) tail. Translation and mRNA turnover
As noted in the previous section, translation and mRNA decay are intimately linked. Additional support for such
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
VOLUME 2 | APRIL 2001 | 2 3 9
© 2001 Macmillan Magazines Ltd