The RNA helicase Dhh1p cooperates with Rbp1p to promote porin mRNA decay via its non-conserved C-terminal domain

The yeast RNA helicase Dhh1p has been shown to associate with components of mRNA decay and is involved in mRNA decapping and degradation. An RNA-binding protein, Rbp1p, is known to bind to the 3'-UTR of porin (POR1) mRNA, and induces mRNA decay by an uncharacterized mechanism. Here, we show that Dhh1p can associate with POR1 mRNA and specifically promote POR1 mRNA decay via its interaction with Rbp1p. As compared to its mammalian homolog RCK/p54/DDX6, Dhh1p has a unique and long extension at its C-terminus. Interestingly, this non-conserved C-terminal region of Dhh1p is required for interaction with Rbp1p and modulating Rbp1p-mediated POR1 mRNA decay. Notably, expression of a C-terminal 81-residue deleted Dhh1p can fully complement the growth defect of a dhh1Δ strain and retains its function in regulating the mRNA level of an RNA-binding protein Edc1p. Moreover, mammalian DDX6 became capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1Δ strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the non-conserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.     

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.     

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.     

The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.     

Localization to,and Effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on Stress Granules in Saccharomyces cerevisiae

The regulation of translation and mRNA degradation in eukaryotic cells involves the formation of cytoplasmic mRNP granules referred to as P-bodies and stress granules. The yeast Pbp1 protein and its mammalian ortholog, Ataxin-2, localize to stress granules and promote their formation. In Saccharomyces cerevisiae, Pbp1 also interacts with the Pab1, Lsm12, Pbp4, and Dhh1 proteins. In this work, we determined whether these Pbp1 interacting proteins also accumulated in stress granules and/or could affect their formation. These experiments revealed the following observations. First, the Lsm12, Pbp4, and Dhh1 proteins all accumulate in stress granules, whereas only the Dhh1 protein is a constitutive P-body component. Second, deletion or over-expression of the Pbp4 and Lsm12 proteins did not dramatically affect the formation of stress granules or P-bodies. In contrast, Pbp1 and Dhh1 over-expression inhibits cell growth, and for Dhh1, leads to the accumulation of stress granules. Finally, a strain lacking the Pab1 protein was reduced at forming stress granules, although they could still be detected. This indicates that Pab1 affects, but is not absolutely required for, stress granule formation. These observations offer new insight into the function of stress granule components with roles in stress granule assembly and mRNP regulation.     

General translational repression by activators of mRNA decapping

Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit translation and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing non-translating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation. Strains lacking both Dhh1p and Pat1p show strong defects in mRNA decapping and P body formation and are blocked in translational repression. Contrastingly, overexpression of Dhh1p or Pat1p causes translational repression, P body formation, and arrests cell growth. Dhh1p, and its human homolog, RCK/p54, repress translation in vitro, and Dhh1p function is bypassed in vivo by inhibition of translational initiation. These results identify a broadly acting mechanism of translational repression that targets mRNAs for decapping and functions in translational control. We propose this mechanism is competitively balanced with translation, and shifting this balance is an important basis of translational control.     

The Cth2 ARE-binding protein recruits the Dhh1 helicase to promote the decay of succinate dehydrogenase SDH4 mRNA in response to iron deficiency

Iron is an essential nutrient that participates as a redox co-factor in a broad range of cellular processes. In response to iron deficiency, the budding yeast Saccharomyces cerevisiae induces the expression of the Cth1 and Cth2 mRNA-binding proteins to promote a genome-wide remodeling of cellular metabolism that contributes to the optimal utilization of iron. Cth1 and Cth2 proteins bind to specific AU-rich elements within the 3'-untranslated region of many mRNAs encoding proteins involved in iron-dependent pathways, thereby promoting their degradation. Here, we show that the DEAD box Dhh1 helicase plays a crucial role in the mechanism of Cth2-mediated mRNA turnover. Yeast two-hybrid experiments indicate that Cth2 protein interacts in vivo with the carboxyl-terminal domain of Dhh1. We demonstrate that the degradation of succinate dehydrogenase SDH4 mRNA, a known target of Cth2 on iron-deficient conditions, depends on Dhh1. In addition, we localize the Cth2 protein to cytoplasmic processing bodies in strains defective in the 5' to 3' mRNA decay pathway. Finally, the degradation of trapped SDH4 mRNA intermediates by Cth2 supports the 5' to 3' directionality of mRNA turnover. Taken together, these results suggest that Cth2 protein recruits the Dhh1 helicase to ARE-containing mRNAs to promote mRNA decay.     

Processing bodies are not required for mammalian nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.     

The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae

We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth.     

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.     

Yeast cells lacking 5''-->3'' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5'' cap structure.

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.     

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.