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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.

This study analyzed sequencing and clinical data from the Cancer Genome Atlas (TCGA) and gene expression synthesis, and used Chinese glioma Genome Atlas (CGGA) data for external validation. The expression of DCP2 in normal brain and tumor tissue was compared. We analyzed the clinical and molecular characteristics and prognostic value of DCP2 in glioma. In addition, DCP2 expression levels were evaluated in 30 glioma tissue samples and upregulated in glioma samples compared to normal brain tissue (p < 0.001). Multivariate data analysis from TCGA showed that increased DCP2 expression was an independent risk factor for overall survival and prognosis of glioma patients. As indicated by the analysis of the TCGA data set. The expression level of DCP2 is closely related to tumor immunity, including tumor immune cell infiltration, immune score, and co-expression of multiple immune-related genes. In addition, DCP2 was positively correlated with IL-6 and IL-7. Glioma cell proliferation and invasion were evaluated using cell viability, colony formation, wound healing, and transwell assays.Apoptosis and cell cycle were detected by flow cytometry. DCP2 promoted the proliferation, invasion and migration of glioma cells T98G and U251, inhibited apoptosis and blocked the S phase of the cell cycle. As a result of the altered expression of DCP2, a new prognostic biomarker may be identified that can improve patient survival.These findings suggest DCP2 as a potential biomarker for the prognosis of glioma and a candidate immunotherapy target. [ABSTRACT FROM AUTHOR]

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.

Abstract Background Patients with glioblastoma (GBM) have a poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The DCPS inhibitor RG3039 exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of RG3039 in various preclinical models of GBM. Methods DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of RG3039 was determined using GBM cell lines, patient-derived organoids, and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored. Results DCPS is overexpressed in GBM and is associated with poor survival of patients with GBM. The DCPS inhibitor RG3039 exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, RG3039 downregulated STAT5B expression, thereby suppressing proliferation, survival and colony formation of GBM cells. Conclusions DCPS is a promising target for GBM. Inhibition of DCPS with RG3039 at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, RG3039 warrants further study as a potential GBM therapy.

The site-specific antibody-drug conjugates (ADCs), particularly those utilizing the engineered cysteine in Fc fragments of mAbs (THIOMAB™ antibodies), have emerged as a novel class of biotherapeutics for cancer treatment. The engineered cysteine residues in these antibodies are capped by cysteine or glutathione through a disulfide bond. Prior to conjugation with linker-payloads, these caps need to be removed through a reduction process. However, monitoring the efficiency of the decapping process has been challenging due to the lack of effective analytical methods. Intact reversed-phase liquid chromatography-mass spectrometry and hydrophobic interaction chromatography methods failed to separate decapped and capped intact THIOMAB™ mAbs in our study. Instead the fragmentation of mAbs provided a novel strategy to analyze the decapping effiency. After cleavage using a hinge specific enzyme, the generated Fc fragments with and without cysteine and/or glutathione caps displayed different hydrophobicity and were well separated by RPLC, allowing quantitative determination of the decapping efficiency. Enzymes that cleave both above and below the hinge disulfide bonds were tested. The use of FabALATICA can determine percentages of molecules with 0, 1, and 2 cysteine and/or glutathione caps, respectively, regardless of whether the antibody contains the hinge LALA mutations. On the other hand, FabRICATOR enzyme can only be utilized for antibodies without LALA mutations for the overall decapping percentage and cannot be used to estimate intact antibody each with 0, 1, and 2 caps. Therefore, FabALACTICA cleavage followed by RPLC provides a wider application of monitoring the decapping efficiency of all antibodies with the engineered cysteine in Fc., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

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., Competing Interests: TC, HL, MN, JS, YC, WC, YL, CC No competing interests declared, (© 2024, Chen, Liao et al.)

Abstract The occurrence of NAD+ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD+. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD+ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation.

The cap is a 7-methylguanosine attached to the first messenger RNA (mRNA) nucleotide with a 5'-5' triphosphate bridge. This conserved eukaryotic modification confers stability to the transcripts and is essential for translation initiation. The specific mechanisms that govern transcript cytoplasmic longevity and translatability were always of substantial interest. Multiple works aimed at modeling mRNA decay mechanisms, including the onset of decapping, which is the rate-limiting step of mRNA decay. Additionally, with the recent advances in RNA-based vaccines, the importance of efficient synthesis of fully functional mRNAs has increased. Non-capped mRNAs arising during in vitro transcription are highly immunogenic, and multiple approaches were developed to reduce their levels. Efficient and low-cost methods for elimination of non-capped mRNAs in vitro are therefore essential to basic sciences and to pharmaceutical applications. Here, we present a protocol for heterologous expression and purification of catalytically active recombinant Xrn1 from Thermothelomyces (Myceliophthora) thermophilus (Tt&#95;Xrn1). We also describe protocols needed to verify the enzyme quality., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This scientific review deals with the mechanisms of action of cytoplasmic microRNAs, namely post-transcriptional silencing: recruitment of the DCP1-DCP2 decapping complex and disruption of the interaction of mRNA with ribosomes. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka databases. The authors indicate that the key process that determines both mRNA stability and expression efficiency is the removal of the 5’-terminal cap. Decapping of mRNA is controlled by several direct and indirect regulators. The DCP1-DCP2 complex can be recruited directly to mRNA and indirectly with the help of several decapping enhancers: PAT1 directly interacts with DCP1 and the decapping stimulator; EDC, DDX6. It is known that the protein DCP2 (Nudt20) is a representative of the conserved subfamily of Nudix hydrolases, which catalyze the hydrolysis of small nucleotide substrates. It is presented that the DCP1 protein is a small molecule that contains the EVH1 (enabled/vasodilator-stimula­ted phosphoprotein homology 1) domain, which usually acts as a protein-protein interaction module, and a C-terminal trimerization domain. It is known that the DCP1-DCP2 complex exists in an open and closed conformation, with the closed conformation having catalytic activity. DCP2 protein and its enhancer and cofactor partners accumulate in P-bodies. The authors indicate that in P-bodies, 5’-monophosphorylated mRNA is finally cleaved under the action of 5’-3’-exoribonuclease XRN1. XRN exoribonucleases are vital enzymes whose gene deletion is accompanied by intraembryonic lethality against the background of various abnormalities in the development of organs and systems. Thus, recruitment of the DCP1-DCP2 decapping complex and disruption of the interaction of mRNA with ribosomes in the cytoplasm of the cell are mechanisms of post-transcriptional silencing. The stability of the mRNA and the efficiency of expression determines the removal of the 5’ end cap. Termination of translation is caused by mRNA. MicroRNA-mediated degradation of this mRNA can be carried out both in the 3’-5’ and 5’-3’ directions of the mo­lecule.

Recent years have led to the identification of a number of enzymes responsible for RNA decapping. This has provided a basis for further research to identify their role, dependency and substrate specificity. However, the multiplicity of these enzymes and the complexity of their functions require advanced tools to study them. Here, we report a high-throughput fluorescence intensity assay based on RNA aptamers designed as substrates for decapping enzymes. Using a library of differently capped RNA probes we generated a decapping susceptibility heat map, which confirms previously reported substrate specificities of seven tested hydrolases and uncovers novel. We have also demonstrated the utility of our assay for evaluating inhibitors of viral decapping enzymes and performed kinetic studies of the decapping process. The assay may accelerate the characterization of new decapping enzymes, enable high-throughput screening of inhibitors and facilitate the development of molecular tools for a better understanding of RNA degradation pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Abstract NAD is a coenzyme central to metabolism that also serves as a 5′-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5′−3′ exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5′-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3, or Scd6; whereas most of the remaining transcripts utilize nonsense-mediated mRNA decay factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are upregulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

The mRNA 5' cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-containing genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

Oomycete Nudix effectors have characteristics of independent evolution, but adopt a conserved WY-Nudix conformation. Furthermore, multiple oomycete Nudix effectors exhibit mRNA decapping activity., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

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.

Decapping is the enzymatic removal of 5′ cap structures from mRNAs in eukaryotic cells. Cap structures normally enhance mRNA translation and stability, and their excision commits an mRNA to complete 5′–3′ exoribonucleolytic digestion and generally ends the physical and functional cellular presence of the mRNA. Decapping plays a pivotal role in eukaryotic cytoplasmic mRNA turnover and is a critical and highly regulated event in multiple 5′–3′ mRNA decay pathways, including general 5′–3′ decay, nonsense‐mediated mRNA decay (NMD), AU‐rich element‐mediated mRNA decay, microRNA‐mediated gene silencing, and targeted transcript‐specific mRNA decay. In the yeast Saccharomyces cerevisiae, mRNA decapping is carried out by a single Dcp1‐Dcp2 decapping enzyme in concert with the accessory activities of specific regulators commonly known as decapping activators or enhancers. These regulatory proteins include the general decapping activators Edc1, 2, and 3, Dhh1, Scd6, Pat1, and the Lsm1‐7 complex, as well as the NMD‐specific factors, Upf1, 2, and 3. Here, we focus on in vivo mRNA decapping regulation in yeast. We summarize recently uncovered molecular mechanisms that control selective targeting of the yeast decapping enzyme and discuss new roles for specific decapping activators in controlling decapping enzyme targeting, assembly of target‐specific decapping complexes, and the monitoring of mRNA translation. Further, we discuss the kinetic contribution of mRNA decapping for overall decay of different substrate mRNAs and highlight experimental evidence pointing to the functional coordination and physical coupling between events in mRNA deadenylation, decapping, and 5′–3′ exoribonucleolytic decay. [ABSTRACT FROM AUTHOR]

Changes in the 5' leader of an mRNA can have profound effects on its translational efficiency with little effect on abundance. Sequencing-based methods to accurately map the 5' leader by identifying the first transcribed nucleotide rely on enzymatic removal of the 5' eukaryotic cap structure by tobacco acid pyrophosphatase (TAP). However, commercial TAP production has been problematic and has now been discontinued. RppH, a bacterial enzyme that can also cleave the 5' cap, and Cap-Clip, a plant-derived enzyme, have been marketed as TAP replacements. We have engineered a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme that functions as a superior TAP replacement. It can be purified from E. coli overexpression in high yields using standard biochemical methods. This constitutively active enzyme is four orders of magnitude more catalytically efficient than RppH at 5' cap removal, compares favorably to Cap-Clip, and the 5' monophosphorylated RNA product is suitable for standard RNA cloning methods. This engineered enzyme is a better replacement for TAP treatment than the current marketed use of RppH and can be produced cost-effectively in a general laboratory setting, unlike Cap-Clip.

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., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Decapping is a crucial step in mRNA degradation in eucaryotes and requires the formation of a holoenzyme complex between the decapping enzyme DECAPPING 2 (DCP2) and the decapping enhancer DCP1. In Arabidopsis (Arabidopsis thaliana), DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a direct protein partner of DCP1. The function of both DNE1 and decapping is necessary to maintain phyllotaxis, the regularity of organ emergence in the apex. In this study, we combined in vivo mRNA editing, RNA degradome sequencing, transcriptomics, and small RNA-omics to identify targets of DNE1 and study how DNE1 and DCP2 cooperate in controlling mRNA fate. Our data reveal that DNE1 mainly contacts and cleaves mRNAs in the coding sequence and has sequence cleavage preferences. DNE1 targets are also degraded through decapping, and both RNA degradation pathways influence the production of mRNA-derived small interfering RNAs. Finally, we detected mRNA features enriched in DNE1 targets including RNA G-quadruplexes and translated upstream open reading frames. Combining these four complementary high-throughput sequencing strategies greatly expands the range of DNE1 targets and allowed us to build a conceptual framework describing the influence of DNE1 and decapping on mRNA fate. These data will be crucial to unveil the specificity of DNE1 action and understand its importance for developmental patterning. Multi-transcriptomics reveals a large spectrum of mRNAs targeted by the endoribonuclease DNE1 and provides a conceptual framework to understand its importance for mRNA fate. [ABSTRACT FROM AUTHOR]

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions., (© 2024 Vukovic et al.)

Degradation of many yeast mRNAs involves decapping by the Dcp1:Dcp2 complex. Previous studies on decapping activators Edc3 and Scd6 suggested their limited roles in mRNA decay. RNA-seq analysis of mutants lacking one or both proteins revealed that Scd6 and Edc3 have largely redundant activities in targeting numerous mRNAs for degradation that are masked in the single mutants. These transcripts also are frequently targeted by decapping activators Dhh1 and Pat1, and the collective evidence suggests that Scd6/Edc3 act interchangeably to recruit Dhh1 to Dcp2. Ribosome profiling shows that redundancy between Scd6 and Edc3 and their functional interactions with Dhh1 and Pat1 extend to translational repression of particular transcripts, including a cohort of poorly translated mRNAs displaying interdependent regulation by all four factors. Scd6/Edc3 also participate with Dhh1/Pat1 in post-transcriptional repression of proteins required for respiration and catabolism of alternative carbon sources, which are normally expressed only in limiting glucose. Simultaneously eliminating Scd6/Edc3 increases mitochondrial membrane potential and elevates metabolites of the tricarboxylic acid and glyoxylate cycles typically observed only during growth in low glucose. Thus, Scd6/Edc3 act redundantly, in parallel with Dhh1 and in cooperation with Pat1, to adjust gene expression to nutrient availability by controlling mRNA decapping and decay.

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.