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.

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 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 Upf1p, Nmd2p, and Upf3p Regulate the Decapping and Exonucleolytic Degradation of both Nonsense-Containing mRNAs and Wild-Type mRNAs

2001 ◽  
Vol 21 (5) ◽  
pp. 1515-1530 ◽  
Author(s):  
Feng He ◽  
Allan Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Translation Termination ◽  
Mrna Degradation ◽  
Wild Type ◽  
Rapid Degradation ◽  
Yeast Strains ◽  
Nonsense Mediated Mrna Decay ◽  
Exonucleolytic Degradation ◽  
Decapping Enzyme

ABSTRACT In Saccharomyces cerevisiae, rapid degradation of nonsense-containing mRNAs requires the decapping enzyme Dcp1p, the 5′-to-3′ exoribonuclease Xrn1p, and the three nonsense-mediated mRNA decay (NMD) factors, Upf1p, Nmd2p, and Upf3p. To identify specific functions for the NMD factors, we analyzed the mRNA decay phenotypes of yeast strains containing deletions of DCP1 orXRN1 and UPF1, NMD2, or UPF3. Our results indicate that Upf1p, Nmd2p, and Upf3p regulate decapping and exonucleolytic degradation of nonsense-containing mRNAs. In addition, we show that these factors also regulate the same processes in the degradation of wild-type mRNAs. The participation of the NMD factors in general mRNA degradation suggests that they may regulate an aspect of translation termination common to all transcripts.

scholarly journals Sbp1p Affects Translational Repression and Decapping in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (13) ◽  
pp. 5120-5130 ◽  
Author(s):  
Scott P. Segal ◽  
Travis Dunckley ◽  
Roy Parker
Keyword(s):  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Glucose Deprivation ◽  
Decay Rates ◽  
The Body ◽  
Translational Repression ◽  
Mrna Turnover ◽  
Major Pathway ◽  
Body Formation ◽  
Decapping Enzyme

ABSTRACT The relationship between translation and mRNA turnover is critical to the regulation of gene expression. One major pathway for mRNA turnover occurs by deadenylation, which leads to decapping and subsequent 5′-to-3′ degradation of the body of the mRNA. Prior to mRNA decapping, a transcript exits translation and enters P bodies to become a potential decapping substrate. To understand the transition from translation to decapping, it is important to identify the factors involved in this process. In this work, we identify Sbp1p (formerly known as Ssb1p), an abundant RNA binding protein, as a high-copy-number suppressor of a conditional allele in the decapping enzyme. Sbp1p overexpression restores normal decay rates in decapping-defective strains and increases P-body size and number. In addition, Sbp1p promotes translational repression of mRNA during glucose deprivation. Moreover, P-body formation is reduced in strains lacking Sbp1p. Sbp1p acts in conjunction with Dhh1p, as it is required for translational repression and P-body formation in pat1Δ strains under these conditions. These results identify Sbp1p as a new protein that functions in the transition of mRNAs from translation to an mRNP complex destined for decapping.

scholarly journals Arabidopsis mRNA decay landscape arises from specialized RNA decay substrates, decapping-mediated feedback, and redundancy

2018 ◽  
Vol 115 (7) ◽  
pp. E1485-E1494 ◽  
Author(s):  
Reed S. Sorenson ◽  
Malia J. Deshotel ◽  
Katrina Johnson ◽  
Frederick R. Adler ◽  
Leslie E. Sieburth
Keyword(s):  
Mathematical Modeling ◽  
Mrna Decay ◽  
Scaffold Protein ◽  
Vital Role ◽  
Decay Rates ◽  
Mrna Abundance ◽  
Wild Type ◽  
Mrna Decapping ◽  
Normal Phenotype ◽  
Wide Range

The decay of mRNA plays a vital role in modulating mRNA abundance, which, in turn, influences cellular and organismal processes. In plants and metazoans, three distinct pathways carry out the decay of most cytoplasmic mRNAs: The mRNA decapping complex, which requires the scaffold protein VARICOSE (VCS), removes a protective 5′ cap, allowing for 5′ to 3′ decay via EXORIBONUCLEASE4 (XRN4, XRN1 in metazoans and yeast), and both the exosome and SUPPRESSOR OF VCS (SOV)/DIS3L2 degrade RNAs in the 3′ to 5′ direction. However, the unique biological contributions of these three pathways, and whether they degrade specialized sets of transcripts, are unknown. In Arabidopsis, the participation of SOV in RNA homeostasis is also unclear, because Arabidopsis sov mutants have a normal phenotype. We carried out mRNA decay analyses in wild-type, sov, vcs, and vcs sov seedlings, and used a mathematical modeling approach to determine decay rates and quantify gene-specific contributions of VCS and SOV to decay. This analysis revealed that VCS (decapping) contributes to decay of 68% of the transcriptome, and, while it initiates degradation of mRNAs with a wide range of decay rates, it especially contributes to decay of short-lived RNAs. Only a few RNAs were clear SOV substrates in that they decayed more slowly in sov mutants. However, 4,506 RNAs showed VCS-dependent feedback in sov that modulated decay rates, and, by inference, transcription, to maintain RNA abundances, suggesting that these RNAs might also be SOV substrates. This feedback was shown to be independent of siRNA activity.

scholarly journals Two Related Proteins, Edc1p and Edc2p, Stimulate mRNA Decapping in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 27-37 ◽  
Author(s):  
Travis Dunckley ◽  
Morgan Tucker ◽  
Roy Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Control Point ◽  
Mrna Decapping ◽  
Critical Control Point ◽  
Decapping Enzyme ◽  
And Function ◽  
Related Proteins ◽  
The Relationship

Abstract The major mRNA decay pathway in Saccharomyces cerevisiae occurs through deadenylation, decapping, and 5′ to 3′ degradation of the mRNA. Decapping is a critical control point in this decay pathway. Two proteins, Dcp1p and Dcp2p, are required for mRNA decapping in vivo and for the production of active decapping enzyme. To understand the relationship between Dcp1p and Dcp2p, a combination of both genetic and biochemical approaches were used. First, we demonstrated that when Dcp1p is biochemically separated from Dcp2p, Dcp1p was active for decapping. This observation confirmed that Dcp1p is the decapping enzyme and indicated that Dcp2p functions to allow the production of active Dcp1p. We also identified two related proteins that stimulate decapping, Edc1p and Edc2p (Enhancer of mRNA DeCapping). Overexpression of the EDC1 and EDC2 genes suppressed conditional alleles of dcp1 and dcp2, respectively. Moreover, when mRNA decapping was compromised, deletion of the EDC1 and/or EDC2 genes caused significant mRNA decay defects. The Edc1p also co-immunoprecipitated with Dcp1p and Dcp2p. These results indicated that Edc1p and Edc2p interact with the decapping proteins and function to enhance the decapping rate.

scholarly journals Competition between Decapping Complex Formation and Ubiquitin-Mediated Proteasomal Degradation Controls Human Dcp2 Decapping Activity

2015 ◽  
Vol 35 (12) ◽  
pp. 2144-2153 ◽  
Author(s):  
Stacy L. Erickson ◽  
Elizabeth O. Corpuz ◽  
Jeffrey P. Maloy ◽  
Christy Fillman ◽  
Kristofer Webb ◽  
...  
Keyword(s):  
Complex Formation ◽  
Translation Initiation ◽  
Proteasomal Degradation ◽  
Mrna Degradation ◽  
Cellular Level ◽  
Regulatory Domain ◽  
Mrna Decapping ◽  
C Terminus ◽  
Decapping Enzyme ◽  
The One

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

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