Arabidopsis Decapping 5 Is Required for mRNA Decapping,P-Body Formation,and Translational Repression during Postembryonic Development

Eukaryotic processing bodies (P-bodies) are implicated in mRNA storage and mRNA decapping. We previously found that a decapping complex comprising Decapping 1 (DCP1), DCP2, and Varicose in Arabidopsis thaliana is essential for postembryonic development, but the underlying mechanism is poorly understood. Here, we characterized Arabidopsis DCP5, a homolog of human RNA-associated protein 55, as an additional P-body constituent. DCP5 associates with DCP1 and DCP2 and is required for mRNA decapping in vivo. In spite of its association with DCP2, DCP5 has no effect on DCP2 decapping activity in vitro, suggesting that the effect on decapping in vivo is indirect. In knockdown mutant dcp5-1, not only is mRNA decapping compromised, but the size of P-bodies is also significantly decreased. These results indicate that DCP5 is required for P-body formation, which likely facilitates efficient decapping. During wild-type seed germination, mRNAs encoding seed storage proteins (SSPs) are translationally repressed and degraded. By contrast, in dcp5-1, SSP mRNAs are translated, leading to accumulation of their products in germinated seedlings. In vitro experiments using wheat germ extracts confirmed that DCP5 is a translational repressor. Our results showed that DCP5 is required for translational repression and P-body formation and plays an indirect role in mRNA decapping.     

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

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.     

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.     

Components of the Arabidopsis mRNA decapping complex are required for early seedling development

To understand the mechanisms controlling vein patterning in Arabidopsis thaliana, we analyzed two phenotypically similar mutants, varicose (vcs) and trident (tdt). We had previously identified VCS, and recently, human VCS was shown to function in mRNA decapping. Here, we report that TDT encodes the mRNA-decapping enzyme. VCS and TDT function together in small cytoplasmic foci that appear to be processing bodies. To understand the developmental requirements for mRNA decapping, we characterized the vcs and tdt phenotypes. These mutants were small and chlorotic, with severe defects in shoot apical meristem formation and cotyledon vein patterning. Many capped mRNAs accumulated in tdt and vcs mutants, but surprisingly, some mRNAs were specifically depleted. In addition, loss of decapping arrested the decay of some mRNAs, while others showed either modest or no decay defects, suggesting that mRNAs may show specificity for particular decay pathways (3′ to 5′ and 5′ to 3′). Furthermore, the severe block to postembryonic development in vcs and tdt and the accompanying accumulation of embryonic mRNAs indicate that decapping is important for the embryo-to-seedling developmental transition.     

Multiple Nudix family proteins possess mRNA decapping activity

RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins—Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19—have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m⁷GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m⁷GMP and m⁷GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m⁷GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.     

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.     

The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1

An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m(7)G cap structure at the 5' end of the message. Here, we describe the functional characterization of Dhh1, a highly conserved member of the family of DEAD box-containing proteins, as a regulator of mRNA decapping in Saccharomyces cerevisiae. Dhh1 is a cytoplasmic protein and is shown to be in a complex with the mRNA degradation factor Pat1/Mtr1 and with the 5'-3' exoribonuclease Xrn1. Dhh1 specifically affects mRNA turnover in the deadenylation-dependent decay pathway, but does not act on the degradation of nonsense-containing mRNAs. Cells that lack dhh1 accumulate degradation intermediates that have lost their poly(A) tail but contain an intact 5' cap structure, suggesting that Dhh1 is required for efficient decapping in vivo. Furthermore, recombinant Dhh1 is able to stimulate the activity of the purified decapping enzyme Dcp1 in an in vitro decapping assay. We propose that the DEAD box protein Dhh1 regulates the access of the decapping enzyme to the m(7)G cap by modulating the structure at the 5' end of mRNAs.     

The decapping enzyme Dcp1 participates in translation termination through its interaction with the release factor eRF3 in budding yeast

One of the rate-limiting steps in messenger RNA decay pathway is the 5'-cap cleavage of mRNAs, decapping reaction, which is conducted by the protein complex of Dcp1 and Dcp2. We find here that Dcp1p can interact with the release factor eRF3p (Sup35p) in Saccharomyces cerevisiae. Knockout of DCP1 caused not only the accumulation of nonsense mRNAs possibly due to the impaired decapping activity but also the enhancement of the read-through of nonsense codon. To examine the relationship between the two DCP1-knockout phenotypes, we produced DCP1 point mutants that lack the ability to support the translation termination. Interestingly, decapping activity of Dcp1p was still intact, but its interaction with eRF3p was abolished in the DCP1 mutants, indicating that the two functions originated from different entities of Dcp1p. These results suggest that the decapping enzyme Dcp1p may have an additional role in the translation termination through its interaction with eRF3p.     

The C‐terminal α–α superhelix of Pat is required for mRNA decapping in metazoa

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N‐terminal sequence, a proline‐rich region, a Mid domain and a C‐terminal domain (Pat‐C). We show that Pat‐C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P‐rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P‐rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P‐rich region. Structural analysis revealed that Pat‐C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat‐C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.     

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-β CH₂ group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the β-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the β-phosphate with BH₃ or Se, or substituted at either the α-β or β-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m₂^7,2′-OGpp_SpG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m⁷Gpp_BH3pm⁷G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t_1/2 ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.     

Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post-embryonic development

Although decapping is an important process in eukaryotic mRNA turnover, little is known about this process in plants. Here, we identified Arabidopsis thaliana decapping proteins AtDCP1 and AtDCP2 and showed that (I) AtDCP2 is an active decapping enzyme, (II) AtDCP1 interacts with itself, (III) AtDCP1 and AtDCP2 are localized to cytoplasmic foci (putative Arabidopsis processing body), and (IV) AtDCP1 and AtDCP2 are essential for post-embryonic development. Our findings provide new insights into the role of decapping-dependent mRNA turnover.     

P-bodies directly regulate MARF1-mediated mRNA decay in human cells

Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to contain translationally repressed mRNAs, a causative role for P-bodies in regulating mRNA decay has yet to be established. Enhancer of decapping protein 4 (EDC4) is a core P-body component that interacts with multiple mRNA decay factors, including the mRNA decapping (DCP2) and decay (XRN1) enzymes. EDC4 also associates with the RNA endonuclease MARF1, an interaction that antagonizes the decay of MARF1-targeted mRNAs. How EDC4 interacts with MARF1 and how it represses MARF1 activity is unclear. In this study, we show that human MARF1 and XRN1 interact with EDC4 using analogous conserved short linear motifs in a mutually exclusive manner. While the EDC4–MARF1 interaction is required for EDC4 to inhibit MARF1 activity, our data indicate that the interaction with EDC4 alone is not sufficient. Importantly, we show that P-body architecture plays a critical role in antagonizing MARF1-mediated mRNA decay. Taken together, our study suggests that P-bodies can directly regulate mRNA turnover by sequestering an mRNA decay enzyme and preventing it from interfacing with and degrading targeted mRNAs.