Active Site Conformational Dynamics Are Coupled to Catalysis in the mRNA Decapping Enzyme Dcp2

Aglietti, Robin A.; Floor, Stephen N.; McClendon, Chris L.; Jacobson, Matthew P.; Gross, John D.

doi:10.1016/j.str.2013.06.021

Cited by 24 publications

(31 citation statements)

References 64 publications

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“…Immunopurified GFP–DCP2 exhibited decapping activity (Figure 6A, lanes 2–4 and Figure 6B), as reported previously (4,16). This activity was specific because a DCP2 protein variant that carries a glutamine substitution in one of the four catalytic glutamates (E148Q) was strongly impaired (Figure 6A, lanes 5–7 and Figure 6B), as expected (4,9,33). A western blot carried out with the corresponding samples showed that wild-type DCP2 and the E148Q mutant interacted with DCP1 and EDC4 to comparable levels and that equivalent amounts of immunoprecipitated DCP2 were present in the decapping reaction (Figure 6C, lanes 2 and 3).…”

Section: Resultssupporting

confidence: 63%

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

Chang

Bercovich

Loh

et al. 2014

Nucleic Acids Research

108

133

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The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.

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Section: Resultssupporting

confidence: 63%

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

Chang

Bercovich

Loh

et al. 2014

Nucleic Acids Research

108

133

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“…These strains, dubbed dcp2-E153Q-N245 and dcp2-E198Q-N245 , harbor the same dcp2-N245 allele but each also contains one additional function-inactivating mutation in an active site residue of the Dcp2 Nudix domain, i.e., glutamate (E) to glutamine (Q) substitutions at codon positions 153 and 198, respectively. E153 of Dcp2 has been shown to function as a general base during the hydrolysis reaction and E198 is involved in Mg 2+ coordination within the Nudix domain (Aglietti et al, 2013). E153Q and E198Q mutations essentially eliminate the decapping activity of Dcp2 both in vitro and in vivo ( Aglietti et al, 2013, He and Jacobson, 2015a ).…”

Section: Resultsmentioning

confidence: 99%

General decapping activators target different subsets of inefficiently translated mRNAs

Jacobson

Çelik

2018

Preprint

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29The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and 30 Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed 31 the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C- 32 terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative 33 and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and 34 Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of 35 these factors has substantial indirect consequences for genome-wide mRNA expression; and 36 iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally 37 translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our 38 results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and 39 suggest that these factors may monitor mRNA translation and target unique features of 40 individual mRNAs. 41 3 65 decapping of general wild-type mRNAs (Parker, 2012), and NMD-specific regulators (Upf1, 66 Upf2, and Upf3) are required for decapping of nonsense-containing mRNAs (He and 67 Jacobson, 2015b, Nicholson and Muhlemann, 2010). Edc3 manifests the most fastidious 68 substrate specificity, being required for decapping of only the yeast YRA1 pre-mRNA and 69 RPS28B mRNA (He et al., 2014, Dong et al., 2007, Badis et al., 2004. All of these decapping 70 activators are conserved from yeast to humans, but their precise functions in mRNA 71 decapping regulation are largely unknown. The two major functions proposed for yeast 72 decapping activators, translational repression and decapping enzyme activation (Parker, 73 2012, Nissan et al., 2010, Coller and Parker, 2005), are still controversial (Sweet et al., 2012, 74 Arribere et al., 2011). 75 Yeast decapping activators exhibit highly specific interactions with each other and with 76 the decapping enzyme. Pat1 interacts with both Dhh1 and the Lsm1-7 complex (He and 77 Jacobson, 2015a, Sharif et al., 2013, Sharif and Conti, 2013, Nissan et al., 2010, Bouveret et 78 al., 2000, Wu et al., 2014), Upf1, Upf2, and Upf3 interact with each other (He et al., 1997), 79 and Edc3 interacts with Dhh1 (He and Jacobson, 2015a, Sharif et al., 2013). Pat1, Upf1, and 80 Edc3 also interact with specific binding motifs in the large C-terminal domain of Dcp2 (He and 81 Jacobson, 2015a, Harigaya et al., 2010). These interaction data and additional observations 82 led us to propose a new model for regulation of mRNA decapping (He and Jacobson, 2015a) 83 in which different decapping activators form distinct decapping complexes in vivo, each of 84 which has a unique substrate specificity that targets a subset of yeast mRNAs. To test 85 aspects of this model, and to further understand the roles of Pat1, Dhh1, and the Lsm1-7 86 complex in general mRNA decapping we have analyzed the effects of deletions of the PAT1, 87 5 LSM1, or DHH1 genes and the large Dcp2 C-terminal domain on transc...

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“…To determine whether the decreased steady-state accumulation of YRA1 pre-mRNA in edc3Δdcp2Δ cells harboring the dcp2-N300 or N245 allele is directly attributable to increased decapping activity, we generated dcp2-N245 alleles harboring mutations in catalytically important residues, including those functioning as general acid (K135A) or general base (E153Q), or involved in metal-binding (E198Q) (Aglietti et al 2013), and analyzed the effects of these mutations in YRA1 premRNA decay in dcp2Δ and edc3Δdcp2Δ cells. In contrast to the unmodified dcp2-N245 allele, the dcp2-N245 alleles harboring each of these amino acid substitutions failed to promote YRA1 pre-mRNA decay and resulted in the increased accumulation of YRA1 pre-mRNA in both dcp2Δ and edc3Δdcp2Δ cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

Jacobson

2015

RNA

139

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Decapping commits an mRNA to complete degradation and promotes general 5 ′ to 3 ′ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping.

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Active Site Conformational Dynamics Are Coupled to Catalysis in the mRNA Decapping Enzyme Dcp2

Cited by 24 publications

References 64 publications

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

General decapping activators target different subsets of inefficiently translated mRNAs

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

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