Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p-7p complex is a general activator of decapping and also functions in protecting the 3' ends of deadenylated mRNAs from a 3'-trimming reaction. Lsm1p is the unique member of the Lsm1p-7p complex, distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p-7p complex and the ability of the Lsm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p-7p complex to the P-bodies; (iii) mRNA 3'-end protection could be indicative of the binding of the Lsm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3' end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.     

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.     

Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae

The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.     

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.     

Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae

The major mRNA decay pathway in Saccharomyces cerevisiae occurs through deadenylation, decapping, and 5' to 3' degradation of the mRNA. Decapping is a critical control point in this decay pathway. Two proteins, Dcp1p and Dcp2p, are required for mRNA decapping in vivo and for the production of active decapping enzyme. To understand the relationship between Dcp1p and Dcp2p, a combination of both genetic and biochemical approaches were used. First, we demonstrated that when Dcp1p is biochemically separated from Dcp2p, Dcp1p was active for decapping. This observation confirmed that Dcp1p is the decapping enzyme and indicated that Dcp2p functions to allow the production of active Dcp1p. We also identified two related proteins that stimulate decapping, Edc1p and Edc2p (Enhancer of mRNA DeCapping). Overexpression of the EDC1 and EDC2 genes suppressed conditional alleles of dcp1 and dcp2, respectively. Moreover, when mRNA decapping was compromised, deletion of the EDC1 and/or EDC2 genes caused significant mRNA decay defects. The Edc1p also co-immunoprecipitated with Dcp1p and Dcp2p. These results indicated that Edc1p and Edc2p interact with the decapping proteins and function to enhance the decapping rate.     

The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs

Decapping is a critical step in mRNA decay. In the 5'-to-3' mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5' to 3' exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 3'-ends of mRNAs in vivo from trimming, presumably by binding to the 3'-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3'-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions.     

The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes.

A major pathway of mRNA turnover in eukaryotic cells initiates with deadenylation, leading to mRNA decapping and subsequent 5' to 3' exonuclease digestion. We show that a highly conserved member of the DEAD box family of helicases, Dhh1p, stimulates mRNA decapping in yeast. In dhh1delta mutants, mRNAs accumulate as deadenylated, capped species. Dhh1p's effects on decapping only occur on normal messages as nonsense-mediated decay still occurs in dhh1delta mutants. The role of Dhh1p in decapping appears to be direct, as Dhh1p physically interacts with several proteins involved in mRNA decapping including the decapping enzyme Dcp1p, as well as Lsm1p and Pat1p/Mrt1p, which function to enhance the decapping rate. Additional observations suggest Dhh1p functions to coordinate distinct steps in mRNA function and decay. Dhh1p also associates with Pop2p, a subunit of the mRNA deadenylase. In addition, genetic phenotypes suggest that Dhh1p also has a second biological function. Interestingly, Dhh1p homologs in others species function in maternal mRNA storage. This provides a novel link between the mechanisms of decapping and maternal mRNA translational repression.     

A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies

mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed that mRNA is also required for accumulation of mRNA degradation factors in P bodies. In contrast, knockdown of the decapping enzyme Dcp2, which initiates the actual 5' --> 3' mRNA degradation did not abolish P body formation, indicating it first functions after mRNPs have been targeted to these cytoplasmic foci. These data support a model in which mRNPs undergo several successive steps of remodeling and/or 3' trimming until their composition or structural organization promotes their accumulation in P bodies.     

Nuclear pre-mRNA decapping and 5' degradation in yeast require the Lsm2-8p complex

Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)(+) RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5' degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5' capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.     

Yeast Lsm1p-7p/Pat1p deadenylation-dependent mRNA-decapping factors are required for brome mosaic virus genomic RNA translation

Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5' and 3' noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.     

Analysis of P-body assembly in Saccharomyces cerevisiae

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.     

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.     

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.     

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.     

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.