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Edc3 Function in Yeast and Mammals Is Modulated by Interaction with NAD-Related Compounds

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Edc3 Function in Yeast and Mammals Is Modulated by Interaction with NAD-Related Compounds INVESTIGATION Edc3 Function in Yeast and Mammals Is Modulated by Interaction with NAD-Related Compounds Robert W. Walters,*,** Igor A. Shumilin,‡ Je-Hyun Yoon,§ Wladek Minor,‡ and Roy Parker*,†,1 *Department Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, †Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, ‡Department of Molecular Physiology and Biological Physics, University of § Virginia, Charlottesville, Virginia, Laboratory of Cellular and Molecular Biology, National Institutes of Health, Baltimore, Maryland, **Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona ABSTRACT The control of mRNA translation and degradation is mediated in part by a set of proteins that KEYWORDS can inhibit translation and promote decapping, as well as function in the assembly of cytoplasmic mRNP Edc3 granules referred to as processing bodies (P-bodies). The conserved enhancer of mRNA decapping 3 (Edc3) P bodies protein functions to promote both decapping and P-body assembly. Crystal structures of the YjeF_N decapping domain in hEdc3 identified a putative binding site for a small molecule. Structure modeling of the human mRNA decay Edc3 Yjef_N along with other Yjef_N-containing proteins suggests that this molecule is related to NAD(H). We now show human Edc3 directly binds NADH. We also show that human and yeast Edc3 chemically modify NAD in vitro. Mutations that are predicted to disrupt the binding and/or hydrolysis of an NAD- related molecule by yeast and human Edc3 affect the control of mRNA degradation and/or P-body com- position in vivo. This suggests that the interaction of Edc3 with an NAD-related molecule affects its function in the regulation of mRNA translation and degradation and provides a possible mechanism to couple the energetics of the cell to posttranscriptional control. Moreover, this provides a unique example of and lends strength to the postulated connection of metabolites, enzymes, and RNA. Recent insights suggest that cytoplasmic mRNA regulation is more translation initiation factors. mRNAs entering a degradative pathway extensive and disease-relevant than previously appreciated (Morris are bound by the decapping enzyme, Dcp2, its accessory proteins, et al. 2010). After transcription, maturation, and subsequent nuclear and the deadenylation complex (Franks and Lykke-Andersen 2008). export, mRNAs are regulated at the level of mRNA stability, localiza- Concentrations of degradative machinery and its associated mRNAs tion, and/or protein synthesis. mRNAs do not exist naturally as sol- are found in cytoplasmic foci termed processing bodies (P-bodies) itary molecules, but rather as complexes with their associated proteins, (Franks and Lykke-Andersen 2008; Balagopal and Parker 2009). or mRNPs. It is the combination of inherent cis-elements of each A conserved modulator of decapping and P-body formation is the mRNA and its associated bound protein factors that determine enhancer of mRNA decapping 3 (Edc3) protein. Edc3 directly binds mRNA fate. and stimulates the decapping enzyme in vitro (Harigaya et al. 2010; Translation-competent mRNAs possess the canonical 59 7mG-cap Nissan et al. 2010; Fromm et al. 2011). Edc3 also affects the decay and 39 poly(A) tail cis-elements and are associated with ribosomes and rates and/or steady-state levels of multiple mRNAs in vivo. For exam- ple, in yeast Edc3 affects the stability of specific mRNAs (Badis et al. Copyright © 2014 Walters et al. 2004; Dong et al. 2007) and is required for general decapping when doi: 10.1534/g3.114.010470 the decapping enzyme is partially functional (Kshirsagar and Parker Manuscript received January 27, 2014; accepted for publication February 1, 2014; 2004). In addition, microarray data from Drosophila S2 cells (Eulalio published Early Online February 5, 2014. et al. 2007) indicate that Edc3 modulates the level and decay rates of This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/ hundreds of mRNAs. In addition to directly binding and activating by/3.0/), which permits unrestricted use, distribution, and reproduction in any Dcp2 enzymatic activity (Harigaya et al. 2010), Edc3 in yeast and/or medium, provided the original work is properly cited. humans also interacts with Dhh1/Rck, Lsm1, Scd6/Rap55, and Xrn1 Supporting information is available online at http://www.g3journal.org/lookup/ (Nissan et al. 2010; Kshirsagar and Parker 2004; Ling et al. 2008; suppl/doi:10.1534/g3.114.010470/-/DC1 1Corresponding author: University of Colorado, 3415 Colorado Avenue, JSCBB Tritschler et al. 2009) and is thought to play a scaffolding role in B414, Boulder, CO 80303-0596. E-mail: [email protected] assembly of a larger decapping complex (Decker et al. 2007). Volume 4 | April 2014 | 613 Consistent with these results, Edc3 in yeast plays a major role in the (Anantharaman and Aravind 2004). More recently, structural analyses assembly of P-bodies (Decker et al. 2007), although how Edc3 func- have revealed that YjeF_N domains frequently bind to NAD-related tion is modulated remains to be determined. molecules (Shumilin et al. 2012), which is consistent with a report An emerging theme in posttranscriptional gene regulation is a arguing that the yeast YjeF_N domain–containing protein YNL200C potentially widespread connection between metabolism and the regu- functions in NAD(P)(H) repair (Marbaix et al. 2011). lation of mRNA function. This regulatory network has been suggested In this work, we demonstrate that the Edc3 binds NADH and based on the observations that some metabolic enzymes have been mutations in the predicted binding sites affect Edc3 function. Com- identified as binding and being regulated by mRNAs (Castello et al. parative structural modeling of several Yjef_N domains suggests that 2012; Hentze and Preiss 2010; Mitchell et al. 2013). If metabolites are the Edc3 Yjef_N domain binds a NAD(H)-related molecule. Utilizing involved in regulating mRNAs, then one also anticipates that some isothermal titration calorimetry, we found that hEdc3 binds to NADH previously identified RNA-binding proteins may contain binding with physiological range affinity. Moreover, mutations in the putative pockets for metabolites that would influence their function. Strikingly, NADH-binding pocket alter several aspects of Edc3 function in yeast the structure of the YjeF_N domain of human Edc3 (hEdc3) revealed and human cells, including subcellular localization, P-body formation, a putative small molecule–binding pocket. Moreover, the YjeF_N and the control of mRNA degradation. We also observe that purified domain was required for RNA-binding and dimerization of Edc3, Edc3 can modify NAD in vitro, indicating that Edc3 may have enzy- whereas it also influenced regulation of Edc3 target mRNAs (Ling matic activity. This identifies the interaction of Edc3 with NAD(H) as et al. 2008). Based on these observations, we hypothesized that Edc3 important in the regulation of mRNA translation and degradation and binds to a cellular metabolite in a manner that regulates its function, provides a possible mechanism to couple the energetics of the cell to thereby coupling posttranscriptional regulation of gene expression to posttranscriptional control. This also provides an example of a second some aspect of metabolism. line of evidence for the emerging coupling of metabolism to RNA The putative binding pocket of Edc3 overlapped with several control wherein RNA-binding proteins will bind and be regulated YjeF_N domain–containing proteins from several species, including by metabolites. those with demonstrated binding to NADP and NAD (Ling et al. 2008). Underscoring the similarities between these binding pockets MATERIALS AND METHODS is the common presence of negatively charged invariant residues (Ling et al. 2008; Shumilin et al. 2012). YjeF_N domains are one of the most Plasmids common functionally uncharacterized protein domains (Finn et al. Edc3-mCherry has been described (pRP1574) (Buchan et al. 2008). 2008). They are predicted to possess conserved enzymatic activity yEdc3 mCherry variants were constructed using oRP 1536 and 1537 Figure 1 Modeling of NAD-related compounds bound to various Yjef_N domains. (A) A surface of Tm0922 subunit with bound NAD (PDB:3RSG; only Yjef_N domain is shown). (B) A surface of apo-Tm0922 subunit (PDB:2AX3; only Yjef_N domain is shown) with NAD superimposed from (A). (C) A surface of AI-BP subunit with bound NADP (PDB:3RNO). (D) A surface of apo-AI- BP subunit (PDB:2O8N) with NADP superimposed from (C). A side chain of F162 that blocks the trench is shown in yellow. The left subunit is shown as a surface with the loop b4-aD that blocks the trench compartment colored is shown in yellow. In all panels, the nicotinamide ring of bound NAD(P) disordered or hydrolyzed in the crystal structures is not shown. The carboxylate oxygen atoms of invariant aspartate (D147Tm0922, D188AI-BP) are shown in red in all surface representations. 614 | R. W. Walters et al. (S351L), oRP 1538 and 1539 (K354F), oRP 1540 and 1541 (D466V), Microscopy and the Quikchange II site–direction mutagenesis kit (Stratagene). All microscopy experiments were performed using a Deltavision His-yEdc3 (pRP 1440) has been described (Decker et al. 2007). RT microscope system as described previously (Buchan et al. oRP1708 and oRP1709 were used to clone full-length hEdc3 into 2010). P-bodies were quantified in a blind manner using three pMBP-parallel1 from a cDNA clone. hEdc3-250C has been described independent experiments comprising at least 100 total counted (Ling et al. 2008). oRP1671 and 1672 were used to construct both granules (Buchan et al. 2010). hEdc3-250C-D443V (pRP 2413) and Flag-hEdc3-D443V-RFP (pRP 2414). Flag-hEdc3-RFP was a kind gift from Dr. Mark Larance (Uni- Northern blot analysis versity of Dundee, Dundee, UK) (Larance et al. 2010). Yeast strains, Northern blot analyses were performed using standard methods as plasmids, and oligos used in this study are listed in Supporting previously described (Decker et al. 2007). A standard Student t-test Information, Table S1.
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