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In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

Proteogenomic identification of translated small open reading frames in human has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. One such regulatory microprotein is NBDY, a 68-amino acid component of the human cytoplasmic RNA decapping complex. Heterologously expressed NBDY was previously reported to regulate cytoplasmic ribonucleoprotein (RNP) granules known as P-bodies and reporter gene stability, but the global effect of endogenous NBDY on the cellular transcriptome remained undefined. In this work, we demonstrate that endogenous NBDY directly interacts with the human RNA decapping complex through EDC4 and DCP1A and localizes to P-bodies. Global profiling of RNA stability changes in NBDY knock out (KO) cells reveals dysregulated stability of over 1400 transcripts. DCP2 substrate transcript half-lives are both increased and decreased in NBDY KO cells, which correlates with 5′ UTR length. NBDY deletion additionally alters the stability of non-DCP2 target transcripts, possibly as a result of downregulated expression of nonsense-mediated decay (NMD) factors in NBDY KO cells. We present a comprehensive model of the regulation of RNA stability by NBDY.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

Most eukaryotic mRNAs harbor a characteristic 5′ m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5′-3′ exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1–Dcp2–Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1–Dcp2 holoenzyme.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DCP2 and its interaction with decapping enhancers, including its main partner DCP1. Here, we report that in Arabidopsis, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we find DNE1 predominantly associated with DCP1 but not with DCP2 and reciprocally, suggesting the existence of two distinct protein complexes. We also show that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines leads to growth defects and transcriptomic changes related to the one observed upon inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations, revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation in Arabidopsis. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.One-sentence summaryDNE1, a NYN domain protein interacts with the decapping activator DCP1 and, together with DCP2, specify phyllotactic patterns in Arabidopsis.

In this study, we investigated the significance of a conserved five-amino acid motif ‘AELPR’ in the C-terminal region of helper component–proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVAΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection., Author summary This study revealed that the potyviral helper component proteinase (HCPro) and the host protein VARICOSE (VCS) are linked in a manner that is important for suppression of RNA silencing, formation of potyvirus-induced RNA granules, translation of viral proteins, stability of virions, and development of systemic potato virus A (PVA) infection. The results suggest that HCPro and VCS belong to the core components of large RNP complexes regulating PVA infection. We suggest that these complexes protect viral RNAs in the cytoplasm after release from the replication complex and direct them to translation and intact to the viral particles.

mRNA decay is a key step in regulating the cellular proteome. Cytoplasmic mRNA is largely turned over in processing bodies (P-bodies). P-body units assemble to form P-body granules under stress conditions. How this assembly is regulated, however, remains still poorly understood. Here, we show that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by capturing the Dcp1/2 decapping complex and preventing it from becoming imported into the nucleus by the karyopherin ß Kap95. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a ready releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3 and Scd6. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Scd6 and Edc3 to drive phase separation and hence P-body formation.

Background: The activation of peroxisome proliferator-activated receptor alpha (PPARα) has been shown to reprogram tumor metabolism and exhibits great potential for treating anti-oral tumorigenesis. Methods: In this study, we used a pathway-based strategy to explore possible functional pathways involved in the anticancer activity of PPARα in oral cancer cells through next-generation sequencing (NGS) and bioinformatic approaches. Results: We found that 3919 genes were upregulated and 1060 genes were downregulated through PPARα activation. These genes were mainly involved in the proteasomal, mRNA surveillance, spliceosomal, RNA transport, and RNA degradation pathways, as indicated by GO and KEGG enrichment analysis. Importantly, a total of 13 upregulated genes in the RNA degradation pathway were identified including 3 core exosome factor genes (RRP43, RRP42, and CSL4), 2 TRAMP complex genes (TRF4 and Mtr4), 2 exosome cofactor genes (RRP6 and MPP6), 2 CCR4-NOT complex genes (CNOT2 and CNOT3), 2 Ski complex genes (SKI2 and Ski3), 1 decapping complex gene (EDC4), and 1 gene involved in 5’ exoribonuclease activity (XRN1). Conclusion: Our findings suggest that the activation of PPARα to upregulate the RNA degradation pathway might provide a new strategy for oral cancer treatment.

Cellular mRNA levels are regulated via rates of transcription and decay. Since the removal of the mRNA 5'-cap by the decapping enzyme DCP2 is generally an irreversible step towards decay, it requires regulation. Control of DCP2 activity is likely effected by two interdependent means: by conformational control of the DCP2-DCP1 complex, and by assembly control of the decapping network, an array of mutually interacting effector proteins. Here, we compare three recent and conformationally distinct crystal structures of the DCP2-DCP1 decapping complex in the presence of substrate analogs and decapping enhancers and we discuss alternative substrate recognition modes for the catalytic domain of DCP2. Together with structure-based insight into decapping network assembly, we propose that DCP2-mediated decapping follows more than one path.

14 p.-6 fig., It has long been assumed that the wide reprogramming of gene expression that modulates plant response to unfavorable environmental conditions is mainly controlled at the transcriptional level. A growing body of evidence, however, indicates that posttranscriptional regulatory mechanisms also play a relevant role in this control.Thus, the LSMs, a family of proteins involved in mRNA metabolism highly conserved in eukaryotes,have emerged as prominent regulators of plant tolerance to abiotic stress. Arabidopsis contains two main LSM ring-shaped heteroheptameric complexes,LSM1-7 and LSM2-8, with different subcellular localization and function. The LSM1-7 ring is part of the cytoplasmic decapping complex that regulates mRNA stability.On the other hand, the LSM2-8 complex accumulates in the nucleus to ensure appropriate levels of U6 snRNA and, therefore, correct pre-mRNA splicing. Recent studies reported unexpected results that led to a fundamental change in the assumed consideration that LSM complexes are mere components of the mRNA decapping and splicing cellular machineries. Indeed, these data have demonstrated that LSM1-7 and LSM2-8 rings operate in Arabidopsis by selecting specific RNA targets, depending on the environmental conditions. This specificity allows them to actively imposing particular gene expression patterns that fine-tune plant responses to abiotic stresses.In this review, we will summarize current and past knowledge on the role of LSM rings in modulating plant physiology, with special focus on their function in abiotic stress responses., This work was supported by grant BIO2016-79187-R from AEI/FEDER, UE to JS.

Gene expression is typically quantified as RNA abundance, which is influenced by both synthesis (transcription) and decay. Cytoplasmic decay typically initiates by deadenylation, after which decay can occur through any of three cytoplasmic decay pathways. Recent advances reveal several mechanisms by which RNA decay is regulated to control RNA abundance. mRNA can be post-transcriptionally modified, either indirectly through secondary structure or through direct modifications to the transcript itself, sometimes resulting in subsequent changes in mRNA decay rates. mRNA abundances can also be modified by tapping into pathways normally used for RNA quality control. Regulated mRNA decay can also come about through post-translational modification of decapping complex subunits. Likewise, mRNAs can undergo changes in subcellular localization (for example, the deposition of specific mRNAs into processing bodies, or P-bodies, where stabilization and destabilization occur in a transcript- and context-dependent manner). Additionally, specialized functions of mRNA decay pathways were implicated in a genome-wide mRNA decay analysis in Arabidopsis. Advances made using plants are emphasized in this review, but relevant studies from other model systems that highlight RNA decay mechanisms that may also be conserved in plants are discussed.

Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.

CSDE1 is a new component of miRNA-induced silencing complex and interacts with DCP1–DCP2 decapping machinery with potential roles in miRNA-mediated target degradation., In animals, miRNAs are the most prevalent small non-coding RNA molecules controlling posttranscriptional gene regulation. The Argonaute proteins (AGO) mediate miRNA-guided gene silencing by recruiting multiple factors involved in translational repression, deadenylation, and decapping. Here, we report that CSDE1, an RNA-binding protein linked to stem cell maintenance and metastasis in cancer, interacts with AGO2 within miRNA-induced silencing complex and mediates gene silencing through its N-terminal domains. We show that CSDE1 interacts with LSM14A, a constituent of P-body assembly and further associates to the DCP1–DCP2 decapping complex, suggesting that CSDE1 could promote the decay of miRNA-induced silencing complex-targeted mRNAs. Together, our findings uncover a hitherto unknown mechanism used by CSDE1 in the control of gene expression mediated by the miRNA pathway.

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5′-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC. These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs.

Nonsense-mediated mRNA decay (NMD), a cellular RNA turnover pathway targeting RNAs with features resulting in aberrant translation termination, has recently been found to restrict the replication of positive-stranded RNA ((+)RNA) viruses. As for every other antiviral immune system, there is also evidence of viruses interfering with and modulating NMD to their own advantage. This review will discuss our current understanding of why and how NMD targets viral RNAs, and elaborate counter-defense strategies viruses utilize to escape NMD.

Nonsense-mediated mRNA decay (NMD), a cellular RNA turnover pathway targeting RNAs with features resulting in aberrant translation termination, has recently been found to restrict the replication of positive-stranded RNA((+) RNA) viruses. As for every other antiviral immune system, there is also evidence of viruses interfering with and modulating NMD to their own advantage. This review will discuss our current understanding of why and how NMD targets viral RNAs, and elaborate counter-defense strategies viruses utilize to escape NMD.

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system.

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.

In mammals, nonsense-mediated mRNA decay (NMD) functions in post-transcriptional gene regulation as well as mRNA surveillance. A key NMD factor, Upf1, becomes hyperphosphorylated by SMG1 kinase during the recognition of NMD substrates. Hyperphosphorylated Upf1 interacts with several factors including SMG5, SMG6, SMG7 and PNRC2 to trigger rapid mRNA degradation. However, the possible cross-talk among these factors and their selective use during NMD remain unknown. Here, we show that PNRC2 is preferentially complexed with SMG5, but not with SMG6 or SMG7, and that downregulation of PNRC2 abolishes the interaction between SMG5 and Dcp1a, a component of the decapping complex. In addition, tethering experiments reveal the function of Upf1, SMG5 and PNRC2 at the same step of NMD and the requirement of SMG6 for Upf1 for efficient mRNA degradation. Intriguingly, microarray results reveal the significant overlap of SMG5-dependent NMD substrates more with PNRC2-dependent NMD substrates than with SMG7-dependent NMD substrates, suggesting the functional dominance of SMG5-PNRC2, rather than SMG5-SMG7, under normal conditions. The results provide evidence that, to some extent, endogenous NMD substrates have their own binding preference for Upf1-interacting adaptors or effectors.

The removal of the 5′ 7-methylguanosine mRNA cap structure (decapping) is a central step in the 5′–3′ mRNA degradation pathway and is performed by the Dcp1:Dcp2 decapping complex. The activity of this complex is tightly regulated to prevent premature degradation of the transcript. Here, we establish that the aromatic groove of the EVH1 domain of Schizosaccharomyces pombe Dcp1 can interact with proline-rich sequences in the exonuclease Xrn1, the scaffolding protein Pat1, the helicase Dhh1, and the C-terminal disordered region of Dcp2. We show that this region of Dcp1 can also recruit a previously unidentified enhancer of decapping protein (Edc1) and solved the crystal structure of the complex. NMR relaxation dispersion experiments reveal that the Dcp1 binding site can adopt multiple conformations, thus providing the plasticity that is required to accommodate different ligands. We show that the activator Edc1 makes additional contacts with the regulatory domain of Dcp2 and that an activation motif in Edc1 increases the RNA affinity of Dcp1:Dcp2. Our data support a model where Edc1 stabilizes the RNA in the active site, which results in enhanced decapping rates. In summary, we show that multiple decapping factors, including the Dcp2 C-terminal region, compete with Edc1 for Dcp1 binding. Our data thus reveal a network of interactions that can fine-tune the catalytic activity of the decapping complex.

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

DDX6 is an abundant DEAD-box helicase associated with various complexes involved in mRNA decay and repression. Its interactome in human cells was analyzed to identify its most prominent partners. Among them, three proteins were essential for P-body assembly in all tested conditions: DDX6, 4E-T, and LSM14A., P-bodies are cytoplasmic ribonucleoprotein granules involved in posttranscriptional regulation. DDX6 is a key component of their assembly in human cells. This DEAD-box RNA helicase is known to be associated with various complexes, including the decapping complex, the CPEB repression complex, RISC, and the CCR4/NOT complex. To understand which DDX6 complexes are required for P-body assembly, we analyzed the DDX6 interactome using the tandem-affinity purification methodology coupled to mass spectrometry. Three complexes were prominent: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. The exon junction complex was also found, suggesting DDX6 binding to newly exported mRNAs. Finally, some DDX6 was associated with polysomes, as previously reported in yeast. Despite its high enrichment in P-bodies, most DDX6 is localized out of P-bodies. Of the three complexes, only the decapping and CPEB-like complexes were recruited into P-bodies. Investigation of P-body assembly in various conditions allowed us to distinguish required proteins from those that are dispensable or participate only in specific conditions. Three proteins were required in all tested conditions: DDX6, 4E-T, and LSM14A. These results reveal the variety of pathways of P-body assembly, which all nevertheless share three key factors connecting P-body assembly to repression.

Decapping 1 (DCP1), Decapping 2 (DCP2) and VARICOSE (VCS) are components of the decapping complex that removes the 7-methyl-guanosine 5'-diphosphate from the 5' end of mRNAs. In animals, the decapping proteins are involved in miRNA-mediated gene silencing, whereas in plants the roles of the decapping proteins in the miRNA pathway are not well understood. Here we demonstrated that the accumulation of miRNAs decreased in dcp1, dcp2 and vcs mutants, indicating that DCP1, DCP2 and VCS are important for the miRNA pathway in Arabidopsis thaliana. The primary miRNAs (pri-miRNAs) did not increase and miRNA biogenesis components did not decrease in these mutants, suggesting that the miRNA decrease in decapping mutants is not due to the defect of pri-miRNA processing. We showed that the accumulation of miRNA targets increased concomitantly with the decrease of miRNA in the decapping mutants. Our results suggested that the seedling lethal phenotypes in the dcp1, dcp2 and vcs mutants are caused not only by the defect in decapping, but also by the disruption of miRNA-mediated gene regulation.

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5′ m7G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m7GpppN) and m7GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m7G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m7GpSpppSm7G, inhibited Dcp1/2 20 times more efficiently than m7GpppN or m7GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m7GpSpppSm7G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m7GpSpppSm7G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5′ cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species.

The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3′ end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5′ end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5′ and 3′ mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.

JNK-mediated phosphorylation of the mRNA-decapping protein DCP1a disrupts P body structure, mRNA stability, and gene expression in response to stress and inflammatory stimuli., Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1 treatment transiently increased P body number. Inhibition of TAK1–JNK signaling also affected the number and size of P bodies and the localization of DCP1a, Xrn1, and Edc4. Transcriptome analysis further identified a central role for DCP1a in IL-1–induced messenger ribonucleic acid (mRNA) expression. Phosphomimetic mutation of S315 stabilized IL-8 but not IκBα mRNA, whereas overexpressed DCP1a blocked IL-8 transcription and suppressed p65 NF-κB nuclear activity. Collectively, these data reveal DCP1a as a multifunctional regulator of mRNA expression and suggest a novel mechanism controlling the subcellular localization of DCP1a in response to stress or inflammatory stimuli.

Human cells can fine-tune gene expression in response to a rapidly changing cellular environment by regulating the rates of mRNA decay (28). Although a variety of RNases in eukaryotic cells participate in mRNA turnover, the shortening and removal of the poly(A) tail by the process of deadenylation are frequently the first steps in this process (4, 52, 70). They are carried out by two major cytoplasmic deadenylase complexes, the Pan2-Pan3 and Ccr4-Caf1-Not complexes (52, 59, 70). Deadenylation generally leads to translational silencing of the mRNA, after which the mRNA can either be stored for later use or subjected to rapid decay (23, 25, 63). In the case of deadenylated mRNAs targeted for decay, the decapping complex removes the cap structure from the 5′ end of the transcript to allow access to the mRNA body by the 5′-to-3′ exonuclease, Xrn1 (28). Alternatively, the exosome, a 3′-to-5′ exonuclease complex, degrades the deadenylated mRNA from the 3′ end (65). mRNA-binding proteins can influence the activity of degradative enzymes on a given message by recognizing cis elements within the transcript. One such sequence is the AU-rich element (ARE) located in the 3′ untranslated region (UTR) of many short-lived mRNAs encoding cytokines, growth factors, proto-oncogenes, and other transiently expressed proteins (11, 15, 36, 72). A number of ARE-binding proteins (AUBPs) that activate or suppress decay have been identified. The protein tristetraprolin (TTP) belongs to a family of CCCH zinc finger AUBPs that trigger rapid mRNA decay by recruiting enzymes involved in mRNA turnover (32, 39, 44). The importance of TTP as a physiological regulator of gene expression is underscored by TTP knockout mice in which chronic inflammation due to elevated levels of tumor necrosis factor alpha (TNF-α) protein has been directly attributed to the increased stability of TNF-α mRNA in TTP−/− macrophages (10). Several cell signaling pathways are known to modulate mRNA decay, but it is poorly understood how these signals are communicated to the mRNA decay machinery. Activation of the p38 mitogen-activated protein kinase (MAPK) pathway by a number of external stresses has been shown to impair the deadenylation of ARE mRNA in vivo (18, 67, 68), although the mechanism by which deadenylation is regulated remains unknown. It has been proposed that AUBPs such as TTP can act as adaptor proteins to communicate signaling events to the mRNA decay machinery (8, 45, 54). Phosphorylation of TTP by the p38 MAPK-activated kinase MK2 has been associated with increased stability of ARE-containing transcripts (45, 58, 60). However, as several AUBPs can bind the ARE and exert synergistic or opposing effects in response to the activation of various signaling pathways, it can be difficult to determine the precise role of phosphorylation of TTP by MK2 in ARE mRNA decay. Phosphorylation of TTP by MK2 creates a substrate for the phospho-serine/threonine binding protein 14-3-3 (14, 55). This has been shown to localize TTP to the cytoplasm (35) and to protect TTP from dephosphorylation by protein phosphatase 2A (PP2A) (57). It remains unclear whether 14-3-3 recruitment plays a direct role in inhibiting the activity of phosphorylated TTP. Here we show that TTP in cells can trigger rapid deadenylation and decay of an mRNA to which it is tethered in isolation from other AUBPs. Using in vitro deadenylation assays, we show that TTP copurifies with a deadenylase activity which specifically deadenylates ARE-containing RNA. Phosphorylation of TTP by the p38-activated kinase MK2 promotes 14-3-3 association and inhibits the ability of TTP to trigger the deadenylation of tethered mRNA in cells by preventing the recruitment of cytoplasmic deadenylases. In contrast, phosphorylation does not affect the ability of TTP to bind mRNA. Taken together, these results suggest that phosphorylated TTP may remain poised to rapidly reactivate mRNA deadenylation and decay upon dephosphorylation.

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S pre-initiation complex. Moreover, while Pat1, Edc3, Dhh1 and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function either to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5'-3' exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.

Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism by which aberrant mRNAs harboring premature termination codons are degraded before translation. However, to date, how NMD machinery recruits the general decay complex to faulty mRNAs and degrades those mRNAs remains unclear. Here we identify human proline-rich nuclear receptor coregulatory protein 2 (PNRC2) as a Upf1- and Dcp1a-interacting protein. Downregulation of PNRC2 abrogates NMD, and artificially tethering PNRC2 downstream of a normal termination codon reduces mRNA abundance. Accordingly, PNRC2 preferentially interacts with hyperphosphorylated Upf1 compared with wild-type Upf1 and triggers movement of hyperphosphorylated Upf1 into processing bodies (P bodies). Our observations suggest that PNRC2 plays an essential role in mammalian NMD, mediating the interaction between the NMD machinery and the decapping complex, so as to target the aberrant mRNA-containing RNPs into P bodies.

mRNA decapping is a critical step in eukaryotic cytoplasmic mRNA turnover. Cytoplasmic mRNA decapping is catalyzed by Dcp2 in conjunction with its coactivator Dcp1 and is stimulated by decapping enhancer proteins. mRNAs associated with the decapping machinery can assemble into cytoplasmic mRNP granules called processing bodies (PBs). Evidence suggests that PB-associated mRNPs are translationally repressed and can be degraded or stored for subsequent translation. However, whether mRNP assembly into a PB is important for translational repression, decapping, or decay has remained controversial. Here, we discuss the regulation of decapping machinery recruitment to specific mRNPs and how their assembly into PBs is governed by the relative rates of translational repression, mRNP multimerization, and mRNA decay.

The removal of the 5′ cap structure by the DCP1–DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all α-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.

Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.

Summary A critical step in mRNA degradation is the removal of the 5′ cap structure, which is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of an S . pombe Dcp1p-Dcp2n complex combined with small-angle X-ray scattering analysis (SAXS) reveals that Dcp2p exists in open and closed conformations, with the closed complex being, or closely resembling, the catalytically more active form. This suggests that a conformational change between these open and closed complexes might control decapping. A bipartite RNA-binding channel containing the catalytic site and Box B motif is identified with a bound ATP located in the catalytic pocket in the closed complex, suggesting possible interactions that facilitate substrate binding. Dcp1 stimulates the activity of Dcp2 by promoting and/or stabilizing the closed complex. Notably, the interface of Dcp1 and Dcp2 is not fully conserved, explaining why the Dcp1-Dcp2 interaction in higher eukaryotes requires an additional factor.

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the “prionlike” domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA–protein complex that then aggregates through multiple specific mechanisms.

Post-transcriptional processes have a central role in the regulation of eukaryotic gene expression. Although it has been known for a long time that these processes are functionally linked, often by the use of common protein factors, it has only recently become apparent that many of these processes are also physically connected. Indeed, proteins that are involved in mRNA degradation, translational repression, mRNA surveillance and RNA-mediated gene silencing, together with their mRNA targets, colocalize within discrete cytoplasmic domains known as P bodies. The available evidence indicates that P bodies are sites where mRNAs that are not being translated accumulate, the information carried by associated proteins and regulatory RNAs is integrated, and their fate - either translation, silencing or decay - is decided.

Maternal mRNAs in oocytes are remarkably stable. In mouse, oocyte maturation triggers a transition from mRNA stability to instability. This transition is a critical event in the oocyte-to-embryo transition in which a differentiated oocyte loses its identity as it is transformed into totipotent blastomeres. We previously demonstrated that phosphorylation of MSY2, an RNA-binding protein, and mobilization of mRNAs encoding the DCP1A-DCP2 decapping complex contribute to maternal mRNA destruction during meiotic maturation. We report here that Cnot7, Cnot6l, and Pan2, key components of deadenylation machinery, are also dormant maternal mRNAs that are recruited during oocyte maturation. Inhibiting the maturation-associated increase in CNOT7 (or CNOT6L) using a small interference RNA approach inhibits mRNA deadenylation, whereas inhibiting the increase in PAN2 has little effect. Reciprocally, expressing CNOT7 (or CNOT6L) in oocytes prevented from resuming meiosis initiates deadenylation of mRNAs. These effects on deadenylation are also observed when the total amount of poly (A) is quantified. Last, inhibiting the increase in CNOT7 protein results in an ∼70% decrease in transcription in 2-cell embryos.

It has long been thought that glucocorticoid receptor (GR) functions as a DNA-binding transcription factor in response to its ligand (a glucocorticoid) and thus regulates various cellular and physiological processes. It is also known that GR can bind not only to DNA but also to mRNA; this observation points to the possible role of GR in mRNA metabolism. Recent data revealed a molecular mechanism by which binding of GR to target mRNA elicits rapid mRNA degradation. GR binds to specific RNA sequences regardless of the presence of a ligand. In the presence of a ligand, however, the mRNA-associated GR can recruit PNRC2 and UPF1, both of which are specific factors involved in nonsense-mediated mRNA decay (NMD). PNRC2 then recruits the decapping complex, consequently promoting mRNA degradation. This mode of mRNA decay is termed “GR-mediated mRNA decay” (GMD). Further research demonstrated that GMD plays a critical role in chemotaxis of immune cells by targeting CCL2 mRNA. All these observations provide molecular insights into a previously unappreciated function of GR in posttranscriptional regulation of gene expression. [BMB Reports 2015; 48(7): 367-368]

International audience; Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations.

Cytoplasmic processing bodies, or P-bodies, contain a high concentration of enzymes and factors required for mRNA turnover and translational repression. Recent studies provide evidence that the mRNAs silenced by miRNAs are localized to P-bodies for storage or degradation, perhaps in adjacent subcompartments. mRNP remodeling, potentially induced by miRISC or RNA helicase activity, may cause the modification of the translation initiation complex at the 5' end of mRNA, following translational repression and localization to P-bodies. Further remodeling in P-bodies may facilitate access of the decapping complex to the cap structure, thus inducing mRNA degradation. However, with appropriate signals, stored mRNAs in P-bodies could be released and returned to the translational machinery through mechanisms requiring binding of regulatory proteins to the 3' UTR of mRNAs. Here a model is proposed to explain the repression and degradation stages of the mRNAs within PBs. This model includes preservation or disruption of a stable closed loop structure of the mRNAs, compartmentalization in PBs and mRNA escape triggered by additional binding proteins.

mRNA decapping is a central step in eukaryotic mRNA decay that simultaneously shuts down translation initiation and activates mRNA degradation. A major complex responsible for decapping consists of the decapping enzyme Dcp2 in association with decapping enhancers. An important question is how the activity and accumulation of Dcp2 are regulated at the cellular level to ensure the specificity and fidelity of the Dcp2 decapping complex. Here, we show that human Dcp2 levels and activity are controlled by a competition between decapping complex assembly and Dcp2 degradation. This is mediated by a regulatory domain in the Dcp2 C terminus, which, on the one hand, promotes Dcp2 activation via decapping complex formation mediated by the decapping enhancer Hedls and, on the other hand, targets Dcp2 for ubiquitin-mediated proteasomal degradation in the absence of Hedls association. This competition between Dcp2 activation and degradation restricts the accumulation and activity of uncomplexed Dcp2, which may be important for preventing uncontrolled decapping or for regulating Dcp2 levels and activity according to cellular needs.

The decapping enzymes DCP1 and DCP2 are components of a decapping complex that degrades mRNAs. DCP2 is the catalytic core and DCP1 is an auxiliary subunit. It has been assumed that DCP1 and DCP2 are consistently co-localized in cytoplasmic RNA granules called processing bodies (P-bodies). However, it has not been confirmed whether DCP1 and DCP2 co-localize in Arabidopsis thaliana. In this study, we generated DCP1-green fluorescent protein (GFP) and DCP2-GFP transgenic plants that complemented dcp1 and dcp2 mutants, respectively, to see whether localization of DCP2 is identical to that of DCP1. DCP2 was present throughout the cytoplasm, whereas DCP1 formed P-body-like foci. Use of DCP1-GFP/DCP2-red fluorescent protein (RFP) or DCP1-RFP/DCP2-GFP plants showed that heat treatment induced DCP2 assembly into DCP1 foci. In contrast, cold treatment did not induce DCP2 assembly, while the number of DCP1 foci increased. These changes in DCP1 and DCP2 localization during heat and cold treatments occurred without changes in DCP1 and DCP2 protein abundance. Our results show that DCP1 and DCP2 respond differently to environmental changes, indicating that P-bodies have diverse DCP1 and DCP2 proportions depending on environmental conditions. The localization changes of DCP1 and DCP2 may explain how specific mRNAs are degraded during changes in environmental conditions.

The mRNA processing body (P-body) is a cellular structure that regulates gene expression by degrading cytoplasmic mRNA. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding Ge-1, a 1401-amino-acid protein. Ge-1 contains an N-terminal WD40 motif and C-terminal domains characterized by a repeating ψ(X2–3) motif. Ge-1 co-localized with previously identified P-body components, including proteins involved in mRNA decapping (DCP1a and DCP2) and the autoantigen GW182. The Ge-1 C-terminal domain was necessary and sufficient to target the protein to P-bodies. Following exposure of cells to oxidative stress, Ge-1-containing P-bodies were found adjacent to TIA-containing stress granules. During the recovery period, TIA returned to the nucleus while Ge-1-containing P-bodies localized to the perinuclear region. siRNA-mediated knock-down of Ge-1 resulted in loss of P-bodies containing Ge-1, DCP1a, and DCP2. In contrast, Ge-1-containing P-bodies persisted despite knock-down of DCP2. Taken together, the results of this study show that Ge-1 is a central component of P-bodies and suggest that Ge-1 may act prior to the 5′-decapping step in mRNA degradation.

In eukaryotic cells degradation of bulk mRNA in the 5′ to 3′ direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5′ to 3′ exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins that are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional processes we depleted P-body or essential pathway components fromDrosophilacells and analyzed the effects of these depletions on the expression of reporter constructs, allowing us to monitor specifically NMD, RNAi, or miRNA function. We show that the RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. Our analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence inDrosophila.

▪ Abstract Eukaryotic mRNAs are primarily degraded by removal of the 3′ poly(A) tail, followed either by cleavage of the 5′ cap structure (decapping) and 5′->3′ exonucleolytic digestion, or by 3′ to 5′ degradation. mRNA decapping represents a critical step in turnover because this permits the degradation of the mRNA and is a site of numerous control inputs. Recent analyses suggest decapping of an mRNA consists of four central and related events. These include removal, or inactivation, of the poly(A) tail as an inhibitor of decapping, exit from active translation, assembly of a decapping complex on the mRNA, and sequestration of the mRNA into discrete cytoplasmic foci where decapping can occur. Each of these steps is a demonstrated, or potential, site for the regulation of mRNA decay. We discuss the decapping process in the light of these central properties, which also suggest fundamental aspects of cytoplasmic mRNA physiology that connect decapping, translation, and storage of mRNA.