Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F

Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNA degradation. However, the cap also binds the eukaryotic initiation factor (eIF) complex 4F and its associated proteins. Characterisation of the relationship between decapping and interactions involving eIF4F is an essential step towards understanding polysome disassembly and mRNA decay. Three types of observation suggest how changes in the functional status of eIF4F modulate mRNA stability in vivo. First, partial disruption of the interaction between eIF4E and eIF4G, caused by mutations in eIF4E or the presence of the yeast 4E-binding protein p20, stabilised mRNAs. The interactions of eIF4G and p20 with eIF4E may therefore act to modulate the decapping process. Since we also show that the in vitro decapping rate is not directly affected by the nature of the body of the mRNA, this suggests that changes in eIF4F structure could play a role in triggering decapping during mRNA decay. Second, these effects were seen in the absence of extreme changes in global translation rates in the cell, and are therefore relevant to normal mRNA turnover. Third, a truncated form of eIF4E (Delta196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simply controlled by competition with eIF4E, but is subject to switching between molecular states with different levels of access.     

Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs.

The major pathway of eukaryotic mRNA decay involves deadenylation-dependent decapping followed by 5' to 3' exonucleolytic degradation. By examining interactions among mRNA decay factors, the mRNA, and key translation factors, we have identified a critical transition in mRNP organization that leads to decapping and degradation of yeast mRNAs. This transition occurs after deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and association of the decapping activator complex, Lsm1p-7p, which enhances the coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the mRNA. These results define an important rearrangement in mRNP organization and suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and the recruitment of the Lsm1p-7p complex.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

Differential inhibition of mRNA degradation pathways by novel cap analogs

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.     

Crystal structure of Dcp1p and its functional implications in mRNA decapping

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.     

A tale of two termini:: A functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation

A quarter of century following the prediction that mRNAs are translated in a circular form, recent biochemical and genetic evidence has accumulated to support the idea that communication between the termini of an mRNA is necessary to promote translation initiation. The poly(A)-binding protein (PABP) interacts with the cap-associated eukaryotic initiation factor (eIF) 4G (in yeast and plants) and eIF4B (in plants), a functional consequence of which is to increase the affinity of PABP for poly(A) and to increase the affinity of the cap-binding complex, eIF4F (of which eIF4G is a subunit) for the 5′ cap structure. In mammals, PABP interacts with a novel PABP-interacting protein that also binds eIF4A. The interaction between PABP and those initiation factors associated with the 5′ terminus of an mRNA may also explain the role of PABP during mRNA turnover, as it protects the 5′ cap from attack by Dcp1p, the decapping enzyme. Several of those mRNAs that have evolved functional equivalents to a cap or a poly(A) tail nevertheless require a functional interaction between terminal regulatory elements similar to that observed between the 5′ cap and poly(A) tail, suggesting that efficient translation is predicated on communication between largely-separated regulatory elements within an mRNA.     

RNA decapping inside and outside of processing bodies

Decapping is a central step in eukaryotic mRNA turnover. Recent studies have identified several factors involved in catalysis and regulation of decapping. These include the following: an mRNA decapping complex containing the proteins Dcp1 and Dcp2; a nucleolar decapping enzyme, X29, involved in the degradation of U8 snoRNA and perhaps of other capped nuclear RNAs; and a decapping 'scavenger' enzyme, DcpS, that hydrolyzes the cap structure resulting from complete 3'-to-5' degradation of mRNAs by the exosome. Several proteins that stimulate mRNA decapping by the Dcp1:Dcp2 complex co-localize with Dcp1 and Dcp2, together with Xrn1, a 5'-to-3' exonuclease, to structures in the cytoplasm called processing bodies. Recent evidence suggests that the processing bodies may constitute specialized cellular compartments of mRNA turnover, which suggests that mRNA and protein localization may be integral to mRNA decay.     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.     

A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.     

Multiple mRNA decapping enzymes in mammalian cells

Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5' end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover.     

Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities

Nonsense-mediated mRNA decay (NMD) is a mechanism by which cells recognize and degrade mRNAs that prematurely terminate translation. To date, the polarity and enzymology of NMD in mammalian cells is unknown. We show here that downregulating the Dcp2 decapping protein or the PM/Scl100 component of the exosome (1) significantly increases the abundance of steady-state nonsense-containing but not nonsense-free mRNAs, and (2) significantly slows the decay rate of transiently induced nonsense-containing but not nonsense-free mRNA. Downregulating poly(A) ribonuclease (PARN) also increases the abundance of nonsense-containing mRNAs. Furthermore, NMD factors Upf1, Upf2, and Upf3X coimmunopurify with the decapping enzyme Dcp2, the putative 5'-->3' exonuclease Rat1, the proven 5'-->3' exonuclease Xrn1, exosomal components PM/Scl100, Rrp4, and Rrp41, and PARN. From these and other data, we conclude that NMD in mammalian cells degrades mRNAs from both 5' and 3' ends by recruiting decapping and 5'-->3' exonuclease activities as well as deadenylating and 3'-->5' exonuclease activities.     

Control of Cyclin-Dependent Kinase Inhibitor p27 Expression by Cap-Independent Translation

p27 is a key regulator of cell proliferation through inhibition of G(1) cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5' cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5' untranslated region (5'-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5'-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression

PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs. The mechanisms and corepressors involved in PUF repression remain to be fully identified. Here, we investigated the mode of repression by Saccharomyces cerevisiae Puf5p and Puf4p and found that Puf5p specifically requires Eap1p to repress mRNAs, whereas Puf4p does not. Surprisingly, we observed that Eap1p, which is a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) class of translational inhibitors, does not inhibit the efficient polyribosome association of a Puf5p target mRNA. Rather, we found that Eap1p accelerates mRNA degradation by promoting decapping, and the ability of Eap1p to interact with eIF4E facilitates this activity. Deletion of EAP1 dramatically reduces decapping, resulting in accumulation of deadenylated, capped mRNA. In support of this phenotype, Eap1p associates both with Puf5p and the Dhh1p decapping factor. Furthermore, recruitment of Eap1p to downregulated mRNA is mediated by Puf5p. On the basis of these results, we propose that Puf5p promotes decapping by recruiting Eap1p and associated decapping factors to mRNAs. The implication of these findings is that a 4E-BP can repress protein expression by promoting specific mRNA degradation steps in addition to or in lieu of inhibiting translation initiation.