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Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors

2020; Elsevier BV; Volume: 295; Issue: 44 Linguagem: Inglês

10.1074/jbc.ra120.012618

ISSN

1083-351X

Autores

Elizabeth Abshire, Kelsey Hughes, Rucheng Diao, Sarah F. Pearce, Shreekara Gopalakrishna, Raymond C. Trievel, Joanna Rorbach, Peter L. Freddolino, Aaron C. Goldstrohm,

Tópico(s)

Genetics, Aging, and Longevity in Model Organisms

Resumo

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type–specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells. Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type–specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells. Nocturnin (NOCT) is a member of the exonuclease-endonuclease-phosphatase (EEP) superfamily of enzymes and is conserved from insects to vertebrates (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar, 2Baggs J.E. Green C.B. Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.Curr. Biol. 2003; 13 (12573214): 189-19810.1016/S0960-9822(03)00014-9Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 3Blanco A.M. Gomez-Boronat M. Madera D. Valenciano A.I. Alonso-Gomez A.L. Delgado M.J. First evidence of nocturnin in fish: two isoforms in goldfish differentially regulated by feeding.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2017; 314 (29070504): R304-R31210.1152/ajpregu.00241.2017Crossref PubMed Scopus (5) Google Scholar, 4Green C.B. Douris N. Kojima S. Strayer C.A. Fogerty J. Lourim D. Keller S.R. Besharse J.C. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17517647): 9888-989310.1073/pnas.0702448104Crossref PubMed Scopus (174) Google Scholar, 5Grönke S. Bickmeyer I. Wunderlich R. Jäckle H. Kühnlein R.P. Curled encodes the Drosophila homolog of the vertebrate circadian deadenylase Nocturnin.Genetics. 2009; 183 (19581445): 219-23210.1534/genetics.109.105601Crossref PubMed Scopus (23) Google Scholar, 6Hughes K.L. Abshire E.T. Goldstrohm A.C. Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries.RNA Biol. 2018; 15 (30257600): 1255-126710.1080/15476286.2018.1526541Crossref PubMed Scopus (4) Google Scholar). Whereas members of the EEP superfamily act on diverse substrates, ranging from nucleic acids to phospholipids, NOCT is most similar to the CCR4 subclass, which suggested a role in RNA metabolism (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar, 2Baggs J.E. Green C.B. Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.Curr. Biol. 2003; 13 (12573214): 189-19810.1016/S0960-9822(03)00014-9Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 6Hughes K.L. Abshire E.T. Goldstrohm A.C. Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries.RNA Biol. 2018; 15 (30257600): 1255-126710.1080/15476286.2018.1526541Crossref PubMed Scopus (4) Google Scholar). CCR4 enzymes are exoribonucleases that degrade the 3′ polyadenosine tail of mRNAs (i.e. deadenylases) (7Goldstrohm A.C. Wickens M. Multifunctional deadenylase complexes diversify mRNA control.Nat. Rev. Mol. Cell Biol. 2008; 9 (18334997): 337-34410.1038/nrm2370Crossref PubMed Scopus (295) Google Scholar). One feature that distinguishes NOCT from other CCR4 enzymes is its unique N terminus, the function of which was unknown (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar). Analysis of knockout mice has established roles for NOCT in cellular differentiation and metabolism. NOCT was first discovered due to its circadian expression pattern (2Baggs J.E. Green C.B. Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.Curr. Biol. 2003; 13 (12573214): 189-19810.1016/S0960-9822(03)00014-9Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Subsequent work indicated that NOCT is not essential for circadian gene expression or behavior (4Green C.B. Douris N. Kojima S. Strayer C.A. Fogerty J. Lourim D. Keller S.R. Besharse J.C. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17517647): 9888-989310.1073/pnas.0702448104Crossref PubMed Scopus (174) Google Scholar). Instead, knockout of NOCT results in resistance to high-fat diet–induced obesity. NOCT knockout mice exhibit defects in absorption, transport, and storage of fat (4Green C.B. Douris N. Kojima S. Strayer C.A. Fogerty J. Lourim D. Keller S.R. Besharse J.C. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17517647): 9888-989310.1073/pnas.0702448104Crossref PubMed Scopus (174) Google Scholar, 8Douris N. Kojima S. Pan X. Lerch-Gaggl A.F. Duong S.Q. Hussain M.M. Green C.B. Nocturnin regulates circadian trafficking of dietary lipid in intestinal enterocytes.Curr. Biol. 2011; 21 (21820310): 1347-135510.1016/j.cub.2011.07.018Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In addition, NOCT knockout mice have increased bone mass with reduced bone marrow adiposity, indicating that NOCT negatively regulates osteogenesis while promoting adipogenesis (4Green C.B. Douris N. Kojima S. Strayer C.A. Fogerty J. Lourim D. Keller S.R. Besharse J.C. Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17517647): 9888-989310.1073/pnas.0702448104Crossref PubMed Scopus (174) Google Scholar, 9Kawai M. Green C.B. Lecka-Czernik B. Douris N. Gilbert M.R. Kojima S. Ackert-Bicknell C. Garg N. Horowitz M.C. Adamo M.L. Clemmons D.R. Rosen C.J. A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-γ nuclear translocation.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (20498072): 10508-1051310.1073/pnas.1000788107Crossref PubMed Scopus (116) Google Scholar, 10Kawai M. Delany A.M. Green C.B. Adamo M.L. Rosen C.J. Nocturnin suppresses igf1 expression in bone by targeting the 3' untranslated region of igf1 mRNA.Endocrinology. 2010; 151 (20685873): 4861-487010.1210/en.2010-0407Crossref PubMed Scopus (37) Google Scholar). The biological roles of NOCT in humans remain largely unknown, as do its molecular functions. Given its relationship to CCR4-type deadenylases, past effort has focused on the ability of NOCT to degrade RNA substrates. Initial biochemical assays suggested that NOCT could degrade poly(A) RNA in vitro; however, subsequent analyses using pure recombinant NOCT (rNOCT) did not detect cleavage of poly(A) substrate RNAs (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar, 2Baggs J.E. Green C.B. Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.Curr. Biol. 2003; 13 (12573214): 189-19810.1016/S0960-9822(03)00014-9Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Garbarino-Pico E. Niu S. Rollag M.D. Strayer C.A. Besharse J.C. Green C.B. Immediate early response of the circadian polyA ribonuclease nocturnin to two extracellular stimuli.RNA. 2007; 13 (17400819): 745-75510.1261/rna.286507Crossref PubMed Scopus (68) Google Scholar, 12Estrella M.A. Du J. Korennykh A. Crystal structure of human nocturnin catalytic domain.Sci. Rep. 2018; 8 (30389976): 1629410.1038/s41598-018-34615-0Crossref PubMed Scopus (8) Google Scholar). As only a few RNA substrates have been tested, it remains plausible that NOCT acts on specific RNA substrates and/or requires unknown partners or modifications that were missing from the biochemical assays. Multiple approaches provide evidence that NOCT acts on mRNAs in vivo. First, when directed to a reporter mRNA in tethered function assays, NOCT reduced translation and RNA stability (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar). The repressive activity of NOCT depends on the 3′ end of its target mRNA; it can affect poly(A) reporter mRNA but not a derivative with a 3′ triple helix structure (13Brown J.A. Bulkley D. Wang J. Valenstein M.L. Yario T.A. Steitz T.A. Steitz J.A. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix.Nat. Struct. Mol. Biol. 2014; 21 (24952594): 633-64010.1038/nsmb.2844Crossref PubMed Scopus (165) Google Scholar, 14Wilusz J.E. JnBaptiste C.K. Lu L.Y. Kuhn C.D. Joshua-Tor L. Sharp P.A. A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.Genes Dev. 2012; 26 (23073843): 2392-240710.1101/gad.204438.112Crossref PubMed Scopus (303) Google Scholar). Moreover, mutations in conserved residues of the putative active site of NOCT reduced its repressive activity (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar). In these aspects, NOCT exhibits mRNA regulatory activity similar to the CCR4-NOT deadenylase complex (7Goldstrohm A.C. Wickens M. Multifunctional deadenylase complexes diversify mRNA control.Nat. Rev. Mol. Cell Biol. 2008; 9 (18334997): 337-34410.1038/nrm2370Crossref PubMed Scopus (295) Google Scholar, 15Cooke A. Prigge A. Wickens M. Translational repression by deadenylases.J. Biol. Chem. 2010; 285 (20634287): 28506-2851310.1074/jbc.M110.150763Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Waghray S. Williams C. Coon J.J. Wickens M. Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo.RNA. 2015; 21 (26015597): 1335-134510.1261/rna.051565.115Crossref PubMed Scopus (20) Google Scholar). Further evidence in support of NOCT-mediated mRNA regulation comes from knockout mice or RNAi depletion of NOCT in liver or cultured cells, respectively; loss of NOCT altered gene expression at the transcript level (10Kawai M. Delany A.M. Green C.B. Adamo M.L. Rosen C.J. Nocturnin suppresses igf1 expression in bone by targeting the 3' untranslated region of igf1 mRNA.Endocrinology. 2010; 151 (20685873): 4861-487010.1210/en.2010-0407Crossref PubMed Scopus (37) Google Scholar, 17Kojima S. Gendreau K.L. Sher-Chen E.L. Gao P. Green C.B. Changes in poly(A) tail length dynamics from the loss of the circadian deadenylase Nocturnin.Sci. Rep. 2015; 5 (26586468): 1705910.1038/srep17059Crossref PubMed Scopus (23) Google Scholar, 18Stubblefield J.J. Gao P. Kilaru G. Mukadam B. Terrien J. Green C.B. Temporal control of metabolic amplitude by nocturnin.Cell Rep. 2018; 22 (29386110): 1225-123510.1016/j.celrep.2018.01.011Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 19Hee S.W. Tsai S.H. Chang Y.C. Chang C.J. Yu I.S. Lee P.C. Lee W.J. Yun-Chia Chang E. Chuang L.M. The role of nocturnin in early adipogenesis and modulation of systemic insulin resistance in human.Obesity. 2012; 20 (22331129): 1558-156510.1038/oby.2012.37Crossref PubMed Scopus (16) Google Scholar, 20Onder Y. Laothamatas I. Berto S. Sewart K. Kilaru G. Bordieanu B. Stubblefield J.J. Konopka G. Mishra P. Green C.B. The circadian protein nocturnin regulates metabolic adaptation in brown adipose tissue.iScience. 2019; 19 (31357170): 83-9210.1016/j.isci.2019.07.016Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The effect of human NOCT on mRNAs remained unexplored; therefore, in this study, we analyzed its impact on the transcriptome in cultured human cells. The relationship of NOCT to the EEP superfamily suggests that it may also act on other types of phosphorylated substrates. Precedent for this hypothesis is illustrated by the CCR4-type deadenylase PDE12, which hydrolyzes the signaling molecule 2′–5′ oligoadenylate, in addition to poly(A) RNA substrates (21Pearce S.F. Rorbach J. Haute L.V. D'Souza A.R. Rebelo-Guiomar P. Powell C.A. Brierley I. Firth A.E. Minczuk M. Maturation of selected human mitochondrial tRNAs requires deadenylation.Elife. 2017; 6 (28745585)10.7554/eLife.27596Crossref Scopus (40) Google Scholar, 22Rorbach J. Nicholls T.J. Minczuk M. PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria.Nucleic Acids Res. 2011; 39 (21666256): 7750-776310.1093/nar/gkr470Crossref PubMed Scopus (83) Google Scholar, 23Wood E.R. Bledsoe R. Chai J. Daka P. Deng H. Ding Y. Harris-Gurley S. Kryn L.H. Nartey E. Nichols J. Nolte R.T. Prabhu N. Rise C. Sheahan T. Shotwell J.B. et al.The role of phosphodiesterase 12 (PDE12) as a negative regulator of the innate immune response and the discovery of antiviral inhibitors.J. Biol. Chem. 2015; 290 (26055709): 19681-1969610.1074/jbc.M115.653113Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). We previously analyzed the ability of NOCT to act on a library of phosphorylated nucleotides, sugars, and lipids, none of which were substrates for rNOCT (1Abshire E.T. Chasseur J. Bohn J.A. Del Rizzo P.A. Freddolino P.L. Goldstrohm A.C. Trievel R.C. The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.Nucleic Acids Res. 2018; 46 (29860338): 6257-627010.1093/nar/gky412Crossref PubMed Scopus (14) Google Scholar). Interestingly, a recent study reported that purified NOCT can remove the 2′ phosphate of the NADPH cofactors NADPH and NADP+ (24Estrella M.A. Du J. Chen L. Rath S. Prangley E. Chitrakar A. Aoki T. Schedl P. Rabinowitz J. Korennykh A. The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled).Nat. Commun. 2019; 10 (31147539): 236710.1038/s41467-019-10125-zCrossref PubMed Scopus (28) Google Scholar). The intracellular localization of NOCT remains incompletely understood, which affects determination of its molecular function and substrates. Previously, overexpressed NOCT was reported to be either cytoplasmic or perinuclear, whereas endogenous NOCT has been reported to localize to the nucleus or cytoplasm of mouse embryo cells or to the cytoplasm in Xenopus retina (2Baggs J.E. Green C.B. Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.Curr. Biol. 2003; 13 (12573214): 189-19810.1016/S0960-9822(03)00014-9Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Kawai M. Green C.B. Lecka-Czernik B. Douris N. Gilbert M.R. Kojima S. Ackert-Bicknell C. Garg N. Horowitz M.C. Adamo M.L. Clemmons D.R. Rosen C.J. A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-γ nuclear translocation.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (20498072): 10508-1051310.1073/pnas.1000788107Crossref PubMed Scopus (116) Google Scholar, 25Estrella M.A. Du J. Chen L. Rath S. Prangley E. Chitrakar A. Aoki T. Schedl P. Rabinowitz J. Korennykh A. The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled).Biorxiv. 2019; 10.1101/534560Google Scholar, 26Le P.T. Bornstein S.A. Motyl K.J. Tian L. Stubblefield J.J. Hong H.K. Takahashi J.S. Green C.B. Rosen C.J. Guntur A.R. A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.J. Cell. Physiol. 2019; 234: 20228-2023910.1002/jcp.28623Crossref PubMed Scopus (7) Google Scholar). Intriguingly, sequence analysis of the unique N terminus of mouse and human NOCT detected the presence of a putative mitochondrial targeting sequence (MTS) (Fig. 1a) (6Hughes K.L. Abshire E.T. Goldstrohm A.C. Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries.RNA Biol. 2018; 15 (30257600): 1255-126710.1080/15476286.2018.1526541Crossref PubMed Scopus (4) Google Scholar). Moreover, NOCT mRNA possesses two potential translation initiation sites, only one of which would include the MTS, although their utilization has not been determined. In this report, we examined the function and localization of NOCT protein at the levels of mRNA and NAD metabolism. We find that the N terminus of NOCT is necessary and sufficient to confer mitochondrial localization. Our analysis provides evidence for cell– and tissue type–specific control of processing and localization of NOCT protein. By employing a gain-of-function approach with transcriptome-wide quantitation, we observe that the cytoplasmic form of NOCT affects the levels of hundreds of mRNAs. Finally, we observe specific effects of NOCT overexpression on cellular NADPH and NADP+ levels. Together, our data indicate that NOCT can act as a dual-function enzyme that modulates the levels of mRNAs and NADP(H) in a manner specified by its intracellular localization. To analyze NOCT protein expression, we generated an antigen affinity–purified polyclonal antibody. Its specificity was validated by Western blotting in three ways: by detection of 1) overexpressed NOCT (encoding amino acids 1–431) in HEK293 cells and 2) recombinant purified human NOCT(64–431) (Fig. 1b) and 3) by RNAi-mediated depletion of endogenous NOCT protein using two different short hairpin RNAs (Fig. S1). In Western blots, this antibody detected two NOCT protein isoforms (Fig. 1b). The major NOCT band in the HepG2 liver cell line migrates with an apparent molecular mass of ∼55 kDa, which is larger than the predicted 48.2 kDa of full-length, 431-amino acid NOCT (Fig. 1, a and b). The N terminus of NOCT is highly basic (see below), which may contribute to its reduced electrophoretic mobility. This 55-kDa form of NOCT was not evident in HEK293 cell extract. In both HEK293, and HepG2 cells, a ∼41 kDa protein was also detected, albeit with low relative abundance. The presence of two distinct forms of NOCT suggested the potential for differential translation initiation, protein processing, or alternative mRNA processing. Alternative NOCT mRNA isoforms capable of explaining the ∼41-kDa product have not been reported in humans; nor did we detect NOCT isoforms in human RNA-Seq data; therefore, we focused on differential translation initiation and/or protein processing. Examination of the NOCT mRNA suggested the potential for two translation initiation sites: Met-1 and Met-67 (Fig. 1a). Translation typically initiates at the first AUG codon (Met-1) within an optimal Kozak sequence context (5′-GCCRCCAUGG) (27Kozak M. Effects of long 5' leader sequences on initiation by eukaryotic ribosomes in vitro.Gene Expr. 1991; 1 (1820209): 117-125PubMed Google Scholar, 28Kozak M. A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes.Gene Expr. 1991; 1: 111-115PubMed Google Scholar, 29Aitken C.E. Lorsch J.R. A mechanistic overview of translation initiation in eukaryotes.Nat. Struct. Mol. Biol. 2012; 19 (22664984): 568-57610.1038/nsmb.2303Crossref PubMed Scopus (265) Google Scholar). For NOCT, initiation at Met-1 would produce the full-length 431-amino acid protein. However, the sequence context of NOCT Met-1 (5′-CCCGGCAUGU, Kozak consensus match in boldface type) differs from the optimal context, raising the possibility for leaky scanning and initiation at the downstream Met-67 (5′-UGUUCCAUGG) (30Benitez-Cantos M.S. Yordanova M.M. O'Connor P.B.F. Zhdanov A.V. Kovalchuk S.I. Papkovsky D.B. Andreev D.E. Baranov P.V. Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.Genome Res. 2020; 30 (32669370): 974-98410.1101/gr.257352.119Crossref PubMed Scopus (12) Google Scholar). To interrogate use of these initiation sites, we cloned the NOCT cDNA, including its natural 5′ leader sequence and 431-amino acid ORF, into an expression vector. We then mutated either initiation codon from AUG to GGG (M1X or M67G) and transfected the constructs into HEK293 cells. Western blotting analysis indicates that Met-1 is the predominant initiation site, whereas Met-67 is used only if Met-1 is mutated (Fig. 1b). Expression of WT NOCT cDNA (residues 1–431) increased the level of the ∼41-kDa form. NOCT M1X produced the expected 41.3-kDa NOCT protein, consistent with the scanning mode of translation wherein Met-67 becomes the first AUG codon in this mutant construct. Importantly, the NOCT M67G construct also produced the ∼41-kDa protein, indicating that the second AUG is not necessary for production of the ∼41-kDa isoform. Together, these observations support the alternate hypothesis that translation of NOCT initiates at Met-1, producing a pre-protein that is processed into the ∼41-kDa form. The N terminus of NOCT contains a putative mitochondrial targeting sequence (MTS) and cleavage site for the mitochondrial processing peptidase (MPP) (Fig. 1a) (6Hughes K.L. Abshire E.T. Goldstrohm A.C. Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries.RNA Biol. 2018; 15 (30257600): 1255-126710.1080/15476286.2018.1526541Crossref PubMed Scopus (4) Google Scholar, 31Fukasawa Y. Tsuji J. Fu S.C. Tomii K. Horton P. Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.Mol. Cell. Proteomics. 2015; 14 (25670805): 1113-112610.1074/mcp.M114.043083Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). MitoFates and TPpred2 algorithms score the NOCT N terminus MTS probability at 0.94 and 0.98, respectively (31Fukasawa Y. Tsuji J. Fu S.C. Tomii K. Horton P. Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.Mol. Cell. Proteomics. 2015; 14 (25670805): 1113-112610.1074/mcp.M114.043083Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 32Savojardo C. Martelli P.L. Fariselli P. Casadio R. TPpred2: improving the prediction of mitochondrial targeting peptide cleavage sites by exploiting sequence motifs.Bioinformatics. 2014; 30 (24974200): 2973-297410.1093/bioinformatics/btu411Crossref PubMed Scopus (29) Google Scholar, 33Indio V. Martelli P.L. Savojardo C. Fariselli P. Casadio R. The prediction of organelle-targeting peptides in eukaryotic proteins with grammatical-restrained hidden conditional random fields.Bioinformatics. 2013; 29 (23428638): 981-98810.1093/bioinformatics/btt089Crossref PubMed Scopus (14) Google Scholar). MTS sequences typically reside in the first 90 residues of a protein, form amphipathic helices, have a high arginine content, and contain few negatively charged residues (31Fukasawa Y. Tsuji J. Fu S.C. Tomii K. Horton P. Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.Mol. Cell. Proteomics. 2015; 14 (25670805): 1113-112610.1074/mcp.M114.043083Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Consistent with these features, amino acids 2–15 of NOCT are predicted to be an amphipathic α-helix within a 74-amino acid polypeptide that has a pI of 12.1 and a dozen arginine residues (6Hughes K.L. Abshire E.T. Goldstrohm A.C. Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries.RNA Biol. 2018; 15 (30257600): 1255-126710.1080/15476286.2018.1526541Crossref PubMed Scopus (4) Google Scholar). MitoFates also identified a consensus cleavage site for MPP, including the arginine at the −2-position relative to the cleavage site after Leu-74 (Fig. 1a) (31Fukasawa Y. Tsuji J. Fu S.C. Tomii K. Horton P. Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.Mol. Cell. Proteomics. 2015; 14 (25670805): 1113-112610.1074/mcp.M114.043083Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The resulting NOCT product (aa 75–431, 40.4 kDa) would have a molecular weight consistent with the observed ∼41 kDa NOCT band. The MTS in the NOCT pre-protein is predicted to be recognized by the translocase of outer membrane (TOM) resulting in import into the mitochondrial matrix, where it would be cleaved by MPP to produce the ∼41-kDa isoform (34Bauer N.C. Doetsch P.W. Corbett A.H. Mechanisms regulating protein localization.Traffic. 2015; 16 (26172624): 1039-106110.1111/tra.12310Crossref PubMed Scopus (90) Google Scholar). Deletion of NOCT amino acids 2–15 reduced the MitoFates probability score from 0.94 to 0.035. Consistent with this prediction, deletion of the MTS sequence, NOCTΔ(2–15), abrogated processing of NOCT and resulted in accumulation of a ∼55-kDa protein in HEK293 cells (Fig. 1b). As expected, the M67G mutation did not alter expression and processing of this form of NOCT. Together, these results are consistent with initiation at Met-1 followed by MTS-dependent processing of the ∼55-kDa form of NOCT to ∼41 kDa. We corroborated these observations in the HCT116 colon cancer cell line (Fig. 1c). Interestingly, the processing appears to be cell type–specific, as HepG2 liver cells predominantly produce an unprocessed ∼55-kDa form of endogenous NOCT (Fig. 1b). To further examine the processing of NOCT protein, we appended a 3×FLAG (3F) tag to the C terminus of WT NOCT or the deletion of MTS (NOCTΔ(2–15)) or N terminus (NOCTΔ(2–67)) (the 3F tag adds 3 kDa to each protein). Anti-FLAG Western blotting of HEK293 cells shows that full-length NOCT(1–431)-3F was processed to form a ∼44-kDa product, whereas deletion of the MTS in NOCTΔ(2–15)-3F prevented processing, resulting in expression of a ∼58-kDa product (Fig. 1d). A larger deletion of the N-terminal sequence (NOCTΔ(2–67)-3F) produced the expected ∼44-kDa product. We also titrated NOCT expression plasmid and observed that, at higher amounts of transfected plasmid, the precursor ∼58-kDa form becomes evident, along with the corresponding increase in processed ∼44-kDa NOCT (Fig. 1e). Taken together, these observations support the efficient processing of NOCT in HEK293 cells, consistent with MTS-dependent mitochondrial import and subsequent proteolytic cleavage by MPP. We next examined the localization of NOCT by performing subcellular fractionation of HEK293T cells that were transfected with WT NOCT(1–431)-3F (Fig. 2a). Cell homogenates were separated into cytoplasmic and mitochondrial fractions, and NOCT-3F was detected by Western blotting. The ∼44-kDa form of NOCT-3F was only detected in the mitochondrial fraction, consistent with MTS-dependent import and processing. As expected, cytosolic uL1 ribosome protein and nuclear histone H3 proteins were not enriched in the mitochondrial fraction. The mitochondrial fractions were subsequently treated with proteinase K to degrade proteins that are outside of intact mitochondria. Under these conditions, outer mitochondrial membrane proteins mitofusin 2 and TOM20 undergo proteolysis due to their exposure to proteinase K, whereas proteins in the mitochondrial matrix (uL4m and uS15m subunits of the mitochondrial ribosome) a

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