Identification of Edc3p as an Enhancer of mRNA Decapping in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 729-739
Author(s):  
Meenakshi Kshirsagar ◽  
Roy Parker
Keyword(s):  
Mrna Decay ◽  
Temperature Sensitive ◽  
Early Step ◽  
Major Pathway ◽  
Mrna Decapping ◽  
P Bodies ◽  
Exonuclease Digestion ◽  
Decapping Enzyme ◽  
Two Hybrid ◽  
New Protein

Abstract The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5′-3′ exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5′-3′ degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5′-3′ exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Δ had no effect when combined with the lsm1Δ, dhh1Δ, or pat1Δ mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.

scholarly journals Suppressors of mRNA Decapping Defects Restore Growth Without Major Effects on mRNA Decay Rates or Abundance

Genetics ◽  
2020 ◽  
Vol 216 (4) ◽  
pp. 1051-1069
Author(s):  
Minseon Kim ◽  
Ambro van Hoof
Keyword(s):  
Mrna Decay ◽  
Slow Growth ◽  
Mrna Degradation ◽  
Decay Rates ◽  
Major Pathway ◽  
Mrna Decapping ◽  
Link Type ◽  
Suppression Mechanism ◽  
Decapping Enzyme ◽  
Insight Into

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.

scholarly journals Suppressors of mRNA decapping defects isolated by experimental evolution ameliorate transcriptome disruption without restoring mRNA decay

2020 ◽  
Author(s):  
Minseon Kim ◽  
Ambro van Hoof
Keyword(s):  
Gene Expression ◽  
Experimental Evolution ◽  
Mrna Decay ◽  
Mrna Degradation ◽  
Catalytic Subunit ◽  
Major Pathway ◽  
Continuous Growth ◽  
Mrna Decapping ◽  
Decapping Enzyme ◽  
Insight Into

ABSTRACTFaithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. While the mechanism and regulation of mRNA decay is well-understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here we clarify that Dcp2 is essential for continuous growth and use experimental evolution to identify suppressors of this essentiality. We show that null mutations in at least three different are each sufficient to restore viability to a dcp2Δ, of which kap123Δ and tl(gag)gΔ appear the most specific. Unlike previously reported suppressors of decapping defects, these suppressor do not restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting transcriptome homeostasis. These effects are not limited to mRNAs, but extend to ncRNAs including snoRNAs and XUTs. These results provide important new insight into the importance of decapping and resolves previously conflicting publications about the essentiality of DCP2.

scholarly journals Mutations in trans-acting factors affecting mRNA decapping in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (10) ◽  
pp. 5830-5838 ◽  
Author(s):  
L Hatfield ◽  
C A Beelman ◽  
A Stevens ◽  
R Parker
Keyword(s):  
Mrna Decay ◽  
Decay Rates ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Subsequent Work ◽  
Factors Affecting ◽  
Mrna Decapping ◽  
Specific Effects ◽  
Cell Extracts ◽  
Decapping Enzyme

The decay of several yeast mRNAs occurs by a mechanism in which deadenylation precedes decapping and subsequent 5'-to-3' exonucleolytic decay. In order to identify gene products required for this process of mRNA turnover, we screened a library of temperature-sensitive strains for mutants with altered mRNA degradation. We identified seven mutations in four genes that inhibited mRNA turnover. Two mutations were alleles of the XRN1 5'-to-3' exoribonuclease known to degrade mRNAs following decapping. One mutation defined a new gene, termed DCP1, which in subsequent work was demonstrated to encode a decapping enzyme or a necessary component of a decapping complex. The other mutations defined two additional genes, termed MRT1 and MRT3 (for mRNA turnover). Mutations in the MRT1 and MRT3 genes slow the rate of deadenylation-dependent decapping, show transcript-specific effects on mRNA decay rates, and do not affect the rapid turnover of an mRNA containing an early nonsense codon, which is degraded by a deadenylation-independent decapping mechanism. Importantly, cell extracts from mrt1 and mrt3 strains contain normal levels of the decapping activity required for mRNA decay. These observations suggest that the products of the MRT1 and MRT3 genes function to modulate the rates of decapping that occur following deadenylation.

scholarly journals Detection and Degradation of Nonsense-mediated mRNA Decay Substrates Involve Two Distinct Upf1-bound Complexes

2018 ◽  
Author(s):  
Marine Dehecq ◽  
Laurence Decourty ◽  
Abdelkader Namane ◽  
Caroline Proux ◽  
Joanne Kanaan ◽  
...  
Keyword(s):  
Large Scale ◽  
Mrna Decay ◽  
Affinity Purification ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Telomere Maintenance ◽  
Mrna Decapping ◽  
Nonsense Mediated Mrna Decay ◽  
Decapping Enzyme

AbstractNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

scholarly journals A non-canonical role for the EDC4 decapping factor in regulating MARF1-mediated mRNA decay

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
William R Brothers ◽  
Steven Hebert ◽  
Claudia L Kleinman ◽  
Marc R Fabian
Keyword(s):  
Essential Role ◽  
Mrna Decay ◽  
Rna Binding ◽  
Mrna Turnover ◽  
Core Component ◽  
P Bodies ◽  
Target Mrnas ◽  
Binding Domains ◽  
Bona Fide ◽  
Decapping Enzyme

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.

scholarly journals Temperature-Sensitive Mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 Genes Result in Defects in mRNA Turnover

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 35-47 ◽  
Author(s):  
Dorit Zuk ◽  
Jonathan P Belk ◽  
Allan Jacobson
Keyword(s):  
Mrna Decay ◽  
Trna Synthetase ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Significant Drop ◽  
Nonsense Mediated Decay ◽  
Mrna Stabilization ◽  
Yeast Strains ◽  
Two Hybrid

Abstract In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37°. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.

scholarly journals Analysis of Mutations in the Yeast mRNA Decapping Enzyme

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1273-1285 ◽  
Author(s):  
Sundaresan Tharun ◽  
Roy Parker
Keyword(s):  
Specific Activity ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Alanine Scanning Mutagenesis ◽  
Mrna Decapping ◽  
Nonsense Codons ◽  
Decapping Enzyme ◽  
Decapping Enzymes

Abstract A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5′ to 3′ exonucleolytic degradation. The decapping enzyme that removes the 5′ cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.

scholarly journals DCP2 plays multiple roles during Drosophila development – possible case of moonlighting?

2019 ◽  
Author(s):  
Rohit Kunar ◽  
Jagat K Roy
Keyword(s):  
Protein Expression ◽  
Early Development ◽  
Multiple Roles ◽  
Dorsal Closure ◽  
Larval Brain ◽  
Living World ◽  
Mrna Decapping ◽  
P Bodies ◽  
Active Expression ◽  
Decapping Enzyme

AbstractmRNA decapping proteins (DCPs) are components of the P-bodies in the cell which are hubs of mRNAs targeted for decay and they provide the cell with a reversible pool of mRNAs in response to cellular demands. The Drosophila genome codes for two decapping proteins, DCP1 and DCP2 out of which DCP2 is the cognate decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, we identify active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain. We also demonstrate that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. Our findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.

scholarly journals Caenorhabditis elegans Decapping Proteins: Localization and Functional Analysis of Dcp1, Dcp2, and DcpS during Embryogenesis

2005 ◽  
Vol 16 (12) ◽  
pp. 5880-5890 ◽  
Author(s):  
Sabbi Lall ◽  
Fabio Piano ◽  
Richard E. Davis
Keyword(s):  
Caenorhabditis Elegans ◽  
Posttranscriptional Regulation ◽  
Mrna Decay ◽  
Degradation Pathway ◽  
Obvious Effect ◽  
P Granules ◽  
P Bodies ◽  
Mrna Metabolism ◽  
Decapping Enzyme ◽  
Decapping Enzymes

Though posttranscriptional regulation is important for early embryogenesis, little is understood regarding control of mRNA decay during development. Previous work defined two major pathways by which normal transcripts are degraded in eukaryotes. However it is not known which pathways are key in mRNA decay during early patterning or whether developmental transcripts are turned over via specific pathways. Here we show that Caenorhabditis elegans Dcp2 is localized to distinct foci during embryogenesis, reminiscent of P-bodies, the sites of mRNA degradation in yeast and mammals. However the decapping enzyme of the 3′ to 5′ transcript decay system (DcpS) localizes throughout the cytoplasm, suggesting this degradation pathway is not highly organized. In addition we find that Dcp2 is localized to P-granules, showing that Dcp2 is stored and/or active in these structures. However RNAi of these decapping enzymes has no obvious effect on embryogenesis. In contrast we find that nuclear cap binding proteins (CBP-20 and 80), eIF4G, and PAB-1 are absolutely required for development. Together our data provides further evidence that pathways of general mRNA metabolism can be remarkably organized during development, with two different decapping enzymes localized in distinct cytoplasmic domains.

Sign in / Sign up

Export Citation Format

Share Document