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.     

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.     

Tethering of Poly(A)-binding Protein Interferes with Non-translated mRNA Decay from the 5′ End in Yeast

The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5′ end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5′-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3′ to 5′ mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1–34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1–34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.     

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)‐binding protein (PABP) and the CCR4–NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4–NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4–NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation.     

Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts.

The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.     

dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

The destabilization of AU-rich element (ARE)-containing mRNAs mediated by proteins of the TIS11 family is conserved among eukaryotes including Drosophila. Previous studies have demonstrated that Tristetraprolin, a human protein of the TIS11 family, induces the degradation of ARE-containing mRNAs through a large variety of mechanisms including deadenylation, decapping, and P-body targeting. We have previously shown that the degradation of the mRNA encoding the antimicrobial peptide Cecropin A1 (CecA1) is controlled by the TIS11 protein (dTIS11) in Drosophila cells. In this study, we used CecA1 mRNA as a model to investigate the molecular mechanism of dTIS11-mediated mRNA decay. We observed that during the biphasic deadenylation and decay process of this mRNA, dTIS11 enhances deadenylation performed by the CCR4-CAF-NOT complex while the mRNA is still associated with ribosomes. Sequencing of mRNA degradation intermediates revealed that the complete deadenylation of the mRNA triggers its decapping and decay in both the 5′-3′ and the 3′-5′ directions. Contrary to the observations made for its mammalian homologs, overexpression of dTIS11 does not promote the localization of ARE-containing mRNAs in P-bodies but rather decreases the accumulation of CecA1 mRNA in these structures by enhancing the degradation process. Therefore, our results suggest that proteins of the TIS11 family may have acquired additional functions in the course of evolution from invertebrates to mammals.     

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.     

Pat1 contributes to the RNA binding activity of the Lsm1-7–Pat1 complex

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5′-to-3′ exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7–Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7–Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7–Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7–Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7–Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7–Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.     

HPat a Decapping Activator Interacting with the miRNA Effector Complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.     

Stm1 Modulates mRNA Decay and Dhh1 Function in Saccharomyces cerevisiae

The control of mRNA degradation and translation are important for the regulation of gene expression. mRNA degradation is often initiated by deadenylation, which leads to decapping and 5′–3′ decay. In the budding yeast Saccharomyces cerevisae, decapping is promoted by the Dhh1 and Pat1 proteins, which appear to both inhibit translation initiation and promote decapping. To understand the function of these factors, we identified the ribosome binding protein Stm1 as a multicopy suppressor of the temperature sensitivity of the pat1Δ strain. Stm1 loss-of-function alleles and overexpression strains show several genetic interactions with Pat1 and Dhh1 alleles in a manner consistent with Stm1 working upstream of Dhh1 to promote Dhh1 function. Consistent with Stm1 affecting Dhh1 function, stm1Δ strains are defective in the degradation of the EDC1 and COX17 mRNAs, whose decay is strongly affected by the loss of Dhh1. These results identify Stm1 as an additional component of the mRNA degradation machinery and suggest a possible connection of mRNA decapping to ribosome function.     

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.     

miRISC recruits decapping factors to miRNA targets to enhance their degradation

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5′-to-3′ messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5′ cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.