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The mRNA 5'-cap structure removal by the decapping enzyme DCP2 is a critical step in gene regulation. While DCP2 is the catalytic subunit in the decapping complex, its activity is strongly enhanced by multiple factors, particularly DCP1, which is the major activator in yeast. However, the precise role of DCP1 in metazoans has yet to be fully elucidated. Moreover, in humans, the specific biological functions of the two DCP1 paralogs, DCP1a and DCP1b, remain largely unknown. To investigate the role of human DCP1, we generated cell lines that were deficient in DCP1a, DCP1b, or both to evaluate the importance of DCP1 in the decapping machinery. Our results highlight the importance of human DCP1 in decapping process and show that the EVH1 domain of DCP1 enhances the mRNA-binding affinity of DCP2. Transcriptome and metabolome analyses outline the distinct functions of DCP1a and DCP1b in human cells, regulating specific endogenous mRNA targets and biological processes. Overall, our findings provide insights into the molecular mechanism of human DCP1 in mRNA decapping and shed light on the distinct functions of its paralogs.

Evolutionarily conserved protein associated with topoisomerase II (PAT1) proteins activate mRNA decay through binding mRNA and recruiting decapping factors to optimize posttranscriptional reprogramming. Here, we generated multiple mutants of pat1, pat1 homolog 1 (path1), and pat1 homolog 2 (path2) and discovered that pat triple mutants exhibit extremely stunted growth and all mutants with pat1 exhibit leaf serration while mutants with pat1 and path1 display short petioles. All three PATs can be found localized to processing bodies and all PATs can target ASYMMETRIC LEAVES 2‐LIKE 9 transcripts for decay to finely regulate apical hook and lateral root development. In conclusion, PATs exhibit both specific and redundant functions during different plant growth stages and our observations underpin the selective regulation of the mRNA decay machinery for proper development. [ABSTRACT FROM AUTHOR]

Deadenylation‐dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein–protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'–3' exonuclease XRN1 interact with the enhancer of mRNA‐decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P‐bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4‐XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA‐targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P‐bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P‐bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P‐body dynamics to properly coordinate mRNA decapping with 5′–3′ decay in human cells. Synopsis: Although P‐bodies are highly concentrated in mRNA decay factors, P‐bodies are surprisingly not required for mRNA decay to occur. Here, P‐bodies are shown to inhibit mRNA decapping and to promote mRNA translational inhibition when the 5′–3′ decay pathway is disrupted. Interaction between the 5′–3′ exonuclease XRN1 and the scaffold protein EDC4 couples mRNA decapping with mRNA decay in a human cancer cell line.Altering XRN1‐EDC4 complex stoichiometry generates larger P‐bodies that, in turn, inhibit decapping.P‐bodies support cellular fitness in the absence of XRN1. [ABSTRACT FROM AUTHOR]

The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far. [ABSTRACT FROM AUTHOR]

Summary: To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini).We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild‐type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity.AvrM14 is an extremely selective enzyme capable of removing the protective 5′ cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild‐type AvrM14, but not the catalytically dead version, suppressed immune‐related reactive oxygen species production, altered the abundance of some circadian‐rhythm‐associated mRNA transcripts and reduced the hypersensitive cell‐death response triggered by the flax disease resistance protein M1.To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA‐decapping activity capable of interfering with plant immunity. See also the Commentary on this article by Banfield, 239: 7–9. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered. [ABSTRACT FROM AUTHOR]

Simple Summary: In eukaryotes, the elimination of the protective cap from 5′ end of messenger RNA (mRNA) by the decapping complex is an important mechanism of mRNA decay and translation, affecting several cellular processes. In the nematode C. elegans, perturbations in decapping activity slow development and shorten the lifespan. A microarray analysis performed in mid-aged nematodes with reduced decapping activity revealed a specific role for decapping in the suppression of spermatogenic genes in germ cells during ageing. In addition, the expression of many innate immunity and detoxification genes were upregulated in the somatic cells of the mutant nematodes, despite the lack of pathogens. This upregulation appears to be mediated by the effect of aberrant decapping on both the mRNA stability and the nuclear translocation of the transcription factor PQM-1, which controls the expression of those immunity/detoxification genes. Although the decapping-mediated induction of immunity was found to be detrimental for the normal lifespan, it mitigates the paralysis in a C. elegans model of polyglutamine toxicity. These results reinforce the selectivity of decapping in the regulation of gene expression and provide a link between mRNA decapping and innate immunity in older ages that could serve as a protective mechanism for disturbed proteostasis, commonly associated with human neurodegenerative diseases. Removal of the 5′ cap structure of RNAs (termed decapping) is a pivotal event in the life of cytoplasmic mRNAs mainly catalyzed by a conserved holoenzyme, composed of the catalytic subunit DCP2 and its essential cofactor DCP1. While decapping was initially considered merely a step in the general 5′-3′ mRNA decay, recent data suggest a great degree of selectivity that plays an active role in the post-transcriptional control of gene expression, and regulates multiple biological functions. Studies in Caenorhabditis elegans have shown that old age is accompanied by the accumulation of decapping factors in cytoplasmic RNA granules, and loss of decapping activity shortens the lifespan. However, the link between decapping and ageing remains elusive. Here, we present a comparative microarray study that was aimed to uncover the differences in the transcriptome of mid-aged dcap-1/DCP1 mutant and wild-type nematodes. Our data indicate that DCAP-1 mediates the silencing of spermatogenic genes during late oogenesis, and suppresses the aberrant uprise of immunity gene expression during ageing. The latter is achieved by destabilizing the mRNA that encodes the transcription factor PQM-1 and impairing its nuclear translocation. Failure to exert decapping-mediated control on PQM-1 has a negative impact on the lifespan, but mitigates the toxic effects of polyglutamine expression that are involved in human disease. [ABSTRACT FROM AUTHOR]

Turnip mosaic virus is a devastating potyvirus infecting many economically important brassica crops. In response to this, the plant host engages its RNA silencing machinery, involving AGO proteins, as a prominent strategy to restrain turnip mosaic virus (TuMV) infection. It has also been shown that the mRNA decay components DCP2 and VCS partake in viral infection suppression. Here, we report that the mRNA decapping components LSM1, PAT1, PATH1, and PATH2 are essential for TuMV infection. More specifically, lsm1a/lsm1b double mutants and pat1/path1/path2 triple mutants in summ2 background exhibit resistance to TuMV. Concurrently, we observed that TuMV interferes with the decapping function of LSM1 and PAT proteins as the mRNA-decay target genes UGT87A2 and ASL9 accumulate during TuMV infection. Moreover, as TuMV coat protein can be specifically found in complexes with PAT proteins but not LSM1, this suggests that TuMV “hijacks” decapping components via PAT proteins to support viral infection.[Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Background ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. Results We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. Conclusions ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored.

Gene expression is tightly regulated at the levels of both mRNA translation and stability. The poly(A)‐binding protein (PABP) is thought to play a role in regulating these processes by binding the mRNA 3′ poly(A) tail and interacting with both the translation and mRNA deadenylation machineries. In this study, we directly investigate the impact of PABP on translation and stability of endogenous mRNAs in human cells. Remarkably, our transcriptome‐wide analysis only detects marginal mRNA translation changes in PABP‐depleted cells. In contrast, rapidly depleting PABP alters mRNA abundance and stability, albeit non‐uniformly. Otherwise stable transcripts, including those encoding proteins with constitutive functions, are destabilized in PABP‐depleted cells. In contrast, many unstable mRNAs, including those encoding proteins with regulatory functions, decay at similar rates in presence or absence of PABP. Moreover, PABP depletion‐induced cell death can partially be suppressed by disrupting the mRNA decapping and 5′–3′ decay machinery. Finally, we provide evidence that the LSM1‐7 complex promotes decay of "stable" mRNAs in PABP‐depleted cells. Taken together, these findings suggest that PABP plays an important role in preventing the untimely decay of select mRNA populations. Synopsis: The poly(A) binding protein (PABP) is known to interact with both the translation and mRNA deadenylation machineries. This study finds that PABP plays a critical role in regulating the abundance and stability of select mRNA populations in human cells, but only subtly alters translation efficiency when depleted. Depleting PABP leads to major changes in mRNA abundance with minor changes in translation efficiency.PABP depletion preferentially destabilized mRNAs that contain short untranslated regions, display long half‐lives, are depleted from P‐bodies, and encode proteins with constitutive functions.PABP protects stable transcripts from undergoing deadenylation‐independent decay.The LSM1‐7 complex promotes deadenylation‐independent decay of stable mRNAs in PABP‐depleted cells. [ABSTRACT FROM AUTHOR]

Sepsis is an aberrant systemic inflammatory response mediated by the acute activation of the innate immune system. Neutrophils are important contributors to the innate immune response that controls the infection, but harbour the risk of collateral tissue damage such as thrombosis and organ dysfunction. A better understanding of the modulations of cellular processes in neutrophils and other blood cells during sepsis is needed and can be initiated via transcriptomic profile investigations. To that point, the growing repertoire of publicly accessible transcriptomic datasets serves as a valuable resource for discovering and/or assessing the robustness of biomarkers. We employed systematic literature mining, reductionist approach to gene expression profile and empirical in vitro work to highlight the role of a Nudix hydrolase family member, NUDT16, in sepsis. The relevance and implication of the expression of NUDT16 under septic conditions and the putative functional roles of this enzyme are discussed. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. [ABSTRACT FROM AUTHOR]

Background: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. Results: We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. Conclusions: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored. [ABSTRACT FROM AUTHOR]

mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo , we employed a transgenic rescue strategy to produce Dcp1a -deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a -deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth. • Dcp1a protein expression is developmentally regulated during embryonic growth. • Deletion of the Dcp1a gene by CRISPR/Cas9 leads to embryonic lethality around E10.5. • Dcp1a -deficient embryos exhibit massive growth retardation and cardiac defects. • Transgenic overexpression of human Dcp1a fully rescues the embryonic lethality. [ABSTRACT FROM AUTHOR]

EDC4 is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3’ UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 impairs MARF1 activity by preventing its LOTUS domains from binding target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs.

Exclusively in eukaryotes, turnover of messenger RNA (mRNA) involves the expulsion of methylated-7-guanosine-diphosphate (m7GDP) present at the 5' end, referred to as decapping of mRNA. The decapping event modulates plant developmental processes. Primarily the decapping process is executed by several decapping complex protein factors, of which DCP1, DCP2 and DCP5 are indispensable. In plants, DCP5 participates in proper functioning of processing bodies (P bodies), whereby the aberrant mRNAs are accumulated for decay. The molecular mechanisms underlying the DCP5 mediated processing of mRNAs are appealing. The results of the present investigation showed that DCP5 physically interacts with Ubiquitin 5. The ubiquitin is a 76-amino acid polypeptide that acts as a covalent modifier of innumerable proteins, resulting in cellular homeostasis. Ubiquitin protein initiates the process of ubiquitination resulting in paving path for the proteins to their final destination, protein decay. Future studies may reveal novel role of ubiquitin proteins in the process of mRNA decay. [ABSTRACT FROM AUTHOR]

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

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.

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. [ABSTRACT FROM AUTHOR]

In eukaryotes, the elimination of the m7 GpppN mRNA cap, a process known as decapping, is a critical, largely irreversible and highly regulated step of mRNA decay that withdraws the targeted mRNAs from the pool of translatable templates. The decapping reaction is catalysed by a multiprotein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor, a holoenzyme that is poorly active on its own and needs several accessory proteins (Lsm1-7 complex, Pat1, Edc1-2, Edc3 and/or EDC4) to be fully efficient. Here, we discuss the several crystal structures of Dcp2 domains bound to various partners (proteins or small molecules) determined in the last couple of years that have considerably improved our current understanding of how Dcp2, assisted by its various activators, is recruited to its mRNA targets and adopts its active conformation upon substrate recognition. We also describe how, over the years, elegant integrative structural biology approaches combined to biochemistry and genetics led to the identification of the correct structure of the active Dcp1-Dcp2 holoenzyme among the many available conformations trapped by X-ray crystallography. [ABSTRACT FROM AUTHOR]

Abstract: The LSM domain‐containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P‐body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E‐binding protein 4E‐T and the DEAD‐box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C‐terminal fragment of 4E‐T. The 4E‐T C‐terminus forms a bi‐partite motif that wraps around the N‐terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C‐terminal RecA‐like domain of DDX6. LSM14 binds DDX6 via a unique non‐contiguous motif with distinct directionality as compared to other DDX6‐interacting proteins. Together with mutational and proteomic studies, the LSM14‐DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P‐body assembly. [ABSTRACT FROM AUTHOR]

The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species. [ABSTRACT FROM AUTHOR]

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5' cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA. [ABSTRACT FROM AUTHOR]

Plant acclimation to environmental stresses requires fast signaling to initiate changes in developmental and metabolic responses. Regulation of gene expression by transcription factors and protein kinases acting upstream are important elements of responses to salt and drought. Gene expression can be also controlled at the post-transcriptional level. Recent analyses on mutants in mRNA metabolism factors suggest their contribution to stress signaling. Here we highlight the components of mRNA decay pathways that contribute to responses to osmotic and salt stress. We hypothesize that phosphorylation state of proteins involved in mRNA decapping affect their substrate specificity. [ABSTRACT FROM AUTHOR]

The 5' terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5'-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5'-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA β-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5'-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin complex 1 (mTORC1) is involved in the cellular transcription and translation processes. The undertaken study characterized the enhancer of mRNA decapping protein 4 (Edc4) as mTORC1 interacting protein. Human T lymphoblast (CCRF-CEM) cells were used for mTORC1 purification. Co-immunoprecipitation coupled with immunoblotting analysis was used to confirm the interaction of Edc4 in mTORC1 specific purifications. Further assays were incorporated to conclude the role of mTORC1 in mRNA decapping via Edc4. Edc4 was identified as a new interacting protein with mTORC1 in both the endogenous and myc-tag raptor component mTORC1 specific purifications. Quantitative co-localization using confocal microscopy demonstrated that raptor component of mTORC1 coexists with Edc4 in processing (P) bodies, a site for mRNA degradation. Incubation of cells with rapamycin, a known inhibitor of mTOR kinase activity, increased the total Edc4 protein expression but at the same time decreased the Edc4 interaction with mTORC1. Moreover, rapamycin treatment resulted in a significant decrease in total serine phosphorylated Edc4 protein signal and the total 5'-capped mRNA. These findings provide the first evidence for the pivotal role of mTORC1 in Edc4 regulation. Further in-depth studies are required to get a complete understanding of molecular crosstalk between mTORC1 signaling and mRNA decapping pathway. [ABSTRACT FROM AUTHOR]

Abstract: The African Swine Fever Virus (ASFV) encodes a single Nudix enzyme in its genome, termed the g5R protein (g5Rp). Nudix phosphohydrolases cleave a variety of substrates, such as nucleotides and diphosphoinositol polyphosphates. Previously, ASFV g5Rp was shown to hydrolyze diphosphoinositol polyphosphates and GTP, but was unable to cleave methylated mRNA cap analogues. In vaccinia virus (VACV), a distant relative of ASFV, the D9 and D10 Nudix enzymes were shown to cleave the mRNA cap, but only when the cap was attached to an RNA body. Here, we show that recombinant ASFV g5Rp hydrolyzes the mRNA cap when tethered to an RNA moiety, liberating m7GDP as a product. Mutations in the Nudix motif abolished mRNA decapping activity, confirming that g5Rp was responsible for cap cleavage. The decapping activity of g5Rp was potently inhibited by excess uncapped RNA but not by methylated cap analogues, suggesting that substrate recognition occurs by RNA binding. [Copyright &y& Elsevier]

Describing at a genomic scale how mutations in different genes influence one another is essential to the understanding of how genotype correlates with phenotype and remains a major challenge in biology. Previous studies pointed out the need for accurate measurements of not only synthetic but also buffering inter- actions in the characterization of genetic networks and functional modules. We developed a sensitive and efficient method that allows such measurements at a genomic scale in yeast. In a pilot experiment (41 genome-wide screens), we quantified the fitness of 140,000 double deletion strains relative to the corresponding single mutants and identified many genetic interactions. In addition to synthetic growth defects (validated experimentally with factors newly identified as genetically interfering with mRNA degradation), most of the identified genetic interactions measured weak epistatic effects. These weak effects, rarely meaningful when considered individually, were crucial to defining specific signatures for many gene deletions and had a major contribution in defining clusters of functionally related genes. [ABSTRACT FROM AUTHOR]

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3′-5′ mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG–DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release. [Copyright &y& Elsevier]

Regulation of mRNA decapping is a critical determinant for gene expression. We demonstrate that the poly(A) tail-mediated regulation of mRNA decapping observed in humans can be recapitulated in vitro by the cytoplasmic poly(A)-binding protein PABP through a direct and specific binding to the 5′ end of capped mRNA. The specific association of PABP with the cap occurred only within the context of the RNA whereby a cap attached to an RNA moiety served as the high-affinity substrate but not the cap structure or RNA alone. Binding of PABP to the RNA 5′ end required the presence of the cap and was accentuated by the N7 methyl moiety of the cap. Interestingly, conditions that enhanced hDcp2 decapping activity reduced the affinity of PABP for cap association and consequently its ability to inhibit decapping, suggestive of a regulated association of PABP with the cap. These observations reveal a novel direct involvement of human PABP in the stabilization of mRNA by protecting the 5′ end from decapping. [ABSTRACT FROM AUTHOR]

An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m7G 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 m7G cap by modulating the structure at the 5′ end of mRNAs. [ABSTRACT FROM AUTHOR]

Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors, ∼4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted a-helical segment of the protein. ts1159 also exhibited an ∼30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping. [ABSTRACT FROM AUTHOR]