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Decapping enzymes comprise many members from various organisms, ranging from plants, animals, and viruses. The mechanisms regulating their functions vary and are still largely unknown. Decapping enzymes remove the 5′ cap of eukaryotic mRNA, leading to accelerated RNA decay. They are critical in regulating RNA homeostasis and play essential roles in many cellular and life processes. They are encoded in many organisms and viruses, including vaccinia virus, which was used as the vaccine to eradicate smallpox. Vaccinia virus encodes two decapping enzymes, D9 and D10, that are necessary for efficient viral replication and pathogenesis. However, the underlying molecular mechanisms regulating vaccinia decapping enzymes’ functions are still largely elusive. Here, we demonstrated that vaccinia D10 almost exclusively colocalized with mitochondria. As mitochondria are highly mobile cellular organelles, colocalization of D10 with mitochondria can concentrate D10 locally and mobilize it to efficiently decap mRNAs. Mitochondria were barely observed in “viral factories,” where viral transcripts are produced, suggesting that mitochondrial colocalization provides a spatial mechanism to preferentially decap cellular mRNAs over viral mRNAs. We identified three amino acids at the N terminus of D10 that are required for D10’s mitochondrial colocalization. Loss of mitochondrial colocalization significantly impaired viral replication, reduced D10’s ability to remove the RNA 5′ cap during infection, and diminished D10’s gene expression shutoff and mRNA translation promotion abilities. IMPORTANCE Decapping enzymes comprise many members from various organisms, ranging from plants, animals, and viruses. The mechanisms regulating their functions vary and are still largely unknown. Our study provides evidence that a vaccinia virus-encoded decapping enzyme, D10, colocalizes with mitochondria. Loss of mitochondrial colocalization significantly impairs viral replication, D10’s gene expression shutoff, and mRNA translation promotion ability. Overall, our results suggest that mitochondrial colocalization is a spatial mechanism to concentrate D10 locally and mobilize it to efficiently and preferentially target cellular mRNAs for decapping and promote viral mRNA translation. Our results have broad impacts for understanding the functions and regulatory mechanisms of decapping enzymes.

The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and suggest that these factors may monitor mRNA translation and target unique features of individual mRNAs.

The 5′ messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3′ end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3′ to 5′ degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.

Background Nudix enzymes constitute a family of hydrolases that share a conserved Nudix motif, which catalyzes the hydrolysis of nu cleoside di phosphates linked to another moiety X . Some members are cellular and viral decapping enzymes that hydrolyze the 5´ cap structure on an mRNA molecule. Unlike vaccinia virus, which encodes two Nudix enzymes, orf virus (ORFV) encodes only a single Nudix-containing gene, ORFV071 (OV71). This study investigates the biochemical properties of recombinant OV71 protein and its role in viral replication. Methods In vitro decapping assays using radiolabeled capped RNA substrates were performed to assess OV71 activity in the presence or absence of competitors or metal cations. Electrophoretic mobility shift assays and pulldown assays evaluated the RNA-binding ability of OV71. Decapping-deficient mutant viruses were generated by homologous recombination, and their replication was analyzed using one-step growth curve experiments. Reverse transcription-qPCR quantified host and viral mRNA levels. Results OV71 exhibited intrinsic decapping activity, hydrolyzing long capped RNAs to release m 7 GDP, with optimal activity in the presence of Mn 2+ . It bound both single- and double-stranded RNA and was expressed early during viral replication. Decapping-deficient mutant viruses replicated poorly in cells. Unlike the vaccinia virus decapping-deficient mutant, which triggers host antiviral responses leading to degradation of viral and host mRNAs as well as rRNAs, an orf virus mutant caused accumulation of host-capped RNAs and a severe reduction in viral mRNAs. Notably, host rRNA remained relatively intact compared to wild-type virus infection. Conclusion OV71 is a decapping enzyme that hydrolyzes the cap structure on long capped mRNAs. It binds both single- and double-stranded RNA, suggesting that it may target both RNA species in infected cells. Its decapping activity is critical for efficient orf virus replication. Loss of this activity leads to the accumulation of host-capped mRNAs, a drastic reduction of viral mRNAs, and minimal impact on host rRNAs, indicating a role distinct from that of the vaccinia virus decapping enzymes.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which is a recently discovered enteric coronavirus, is the major aetiological agent that causes severe clinical diarrhoea and intestinal pathological damage in pigs, and it has caused significant economic losses to the swine industry. Nonstructural protein 5, also called 3C-like protease, cleaves viral polypeptides and host immune-related molecules to facilitate viral replication and immune evasion. Here, we demonstrated that SADS-CoV nsp5 significantly inhibits the Sendai virus (SEV)-induced production of IFN-β and inflammatory cytokines. SADS-CoV nsp5 targets and cleaves mRNA-decapping enzyme 1a (DCP1A) via its protease activity to inhibit the IRF3 and NF-κB signaling pathways in order to decrease IFN-β and inflammatory cytokine production. We found that the histidine 41 and cystine 144 residues of SADS-CoV nsp5 are critical for its cleavage activity. Additionally, a form of DCP1A with a mutation in the glutamine 343 residue is resistant to nsp5-mediated cleavage and has a stronger ability to inhibit SADS-CoV infection than wild-type DCP1A. In conclusion, our findings reveal that SADS-CoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by alpha coronaviruses.

The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1δ protein kinase is recruited to cytoplasmic processing bodies (P-bodies) in an evolutionarily conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.

Processing bodies (P-bodies) are one of the most well understood types of RNA granules, and are associated with a variety of diseases, including cancer. mRNA-decapping enzyme 1a (DCP1a), which may be used as a marker to analyze P-bodies, participates in the removal of the 5′-methylguanosine cap from eukaryotic mRNAs as a cofactor. The aim of the present study was to analyze the association between DCP1a expression and clinical features in colorectal carcinoma (CRC). The levels of DCP1a mRNA expression were detected by reverse transcription-quantitative polymerase chain reaction assay in carcinoma and non-carcinoma tissues from 75 patients, while the protein expression levels were evaluated by immunohistochemistry and western blotting. Additional associations between DCP1a expression and clinical characteristics were analyzed by χ2 test and Cox regression analysis. In the 75 cases, the levels of DCP1a mRNA and protein expression were increased in colorectal carcinoma tissues compared with non-carcinoma tissues. A high expression of DCP1a was significantly associated with lower survival rates in patients with CRC compared with patients with low DCP1a expression (P=0.001). Associations with depth of invasion (P=0.008), lymph node metastasis (P=0.001) and tumor node metastasis stage (P=0.001) were also observed. Additional Cox regression analysis revealed that the DCP1a expression (P=0.012) is an independent factor in survival rate. It was also identified that DCP1a may have high expression in colorectal carcinoma tissues and be associated with poor prognosis. This suggests that DCP1a may be a diagnostic marker or prognostic indicator to assist with patient assessments and therapies.

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.

The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and suggest that these factors may monitor mRNA translation and target unique features of individual mRNAs.

Eukaryotic mRNA decapping by Dcp2 is the penultimate step in several mRNA decay pathways. To understand regulation of Dcp2 by ligand interactions, we have assigned the backbone and sidechain methyl Ile (delta1), Leu and Val chemical shifts of the catalytic domain of the S. Cerevisiae enzyme.