The RNA-binding protein 8A (RBM8A)–mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.
BonoFGehringNH. Assembly, disassembly and recycling: the dynamics of exon junction complexes. RNA Biol2011; 8: 24–29.
2.
Le HirHSéraphinB. EJCs at the heart of translational control. Cell2008; 133: 213–6.
3.
Le HirHGatfieldDIzaurraldeEMooreMJ. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. Embo J2001; 20: 4987–97.
4.
SinghGLykke-AndersenJ. New insights into the formation of active nonsense-mediated decay complexes. Trends Biochem Sci2003; 28: 464–6.
5.
Kataoka N, Diem MD, Yoshida M, Hatai C, Dobashi I, Dreyfuss G, Hagiwara M, Ohno M. Specific Y14 domains mediate its nucleo-cytoplasmic shuttling and association with spliced mRNA. Sci Rep 2011;1:92.
6.
GehringNHLamprinakiSKulozikAEHentzeMW. Disassembly of exon junction complexes by PYM. Cell2009; 137: 536–48.
7.
SalicioniAMXiMVanderveerLABalsaraBTestaJRDunbrackRLJrGodwinAK. Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor. Genomics2000; 69: 54–62.
8.
ZhaoXFColaizzo-AnasTNowakNJShowsTBElliottRWAplanPD. The mammalian homologue of mago nashi encodes a serum-inducible protein. Genomics1998; 47: 319–22.
9.
KataokaNDiemMDKimVNYongJDreyfussG. Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. Embo J2001; 20: 6424–33.
10.
LauCKDiemMDDreyfussGVan DuyneGD. Structure of the Y14-Magoh core of the exon junction complex. Curr Biol2003; 13: 933–41.
11.
ChaungTWChaungWLLeeKMTarnWY. The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation. Mol Biol Cell2013; 24: 1–13.
12.
ChaungTWPengPJTarnWY. The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosoimal small nuclear ribonucleoproteins. J Biol Chem2011; 286: 8722–8.
13.
MuromotoRTairaNIkedaOShigaKKamitaniSTogiSKawakamiSSekineYNanboAOritaniKMatsudaT. The exon-junction complex proteins, Y14 and MAGOH regulate STAT3 activation. Biochem Biophys Res Commun2009; 382: 63–68.
14.
OhbayashiNTairaNKawakamiSTogiSSatoNIkedaOKamitaniSMuromotoRSekineYMatsudaT. An RNA biding protein, Y14 interacts with and modulates STAT3 activation. Biochem Biophys Res Commun2008; 372: 475–9.
15.
TogiSShigaKMuromotoRKatoMSomuraYSekineYkonSOritaniKMatsudaT. Y14 positively regulates TNF-α-induced NF-κB transcriptional activity via interacting RIP1 and TRADD beyond an exon junction complex protein. J Immunol2013; 191: 1436–44.
16.
InakiMKatoDUtsugiTOnodaFHanaokaFMurakamiY. Genetic analyses using a mouse cell cycle mutant identifies magoh as a novel gene involved in Cdk regulation. Genes Cells2011; 16: 166–78.
17.
Le HirHGatfieldDBraunICForlerDIzaurraldeE. The protein Mago provides a link between splicing and mRNA localization. Embo Rep2001; 2: 1119–24.
18.
SudoHTsujiABSugyoAKohdaMSogawaCYoshidaCHaradaYNHinoOSagaT. Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model. Genomics2010; 95: 210–6.
19.
SilverDLWatkins-ChowDESchreckKCPierfeliceTJLarsonDMBurnettiAJLiawHJMyungKWalshCAGaianoNPavanWJ. The exon junction complex component Magoh controls brain size by regulating neural stem cell division. Nat Neurosci2010; 13: 551–8.
20.
IshigakiYNakamuraYTatsunoTHashimotoMShimasakiTIwabuchiKTomosugiN. Depletion of RNA-binding protein RBM8A (Y14) causes cell cycle deficiency and apoptosis in human cells. Exp Biol Med2013; 238: 889–97.
21.
IshigakiYNakamuraYTatsunoTHashimotoMIwabuchiKTomosugiN. RNA binding protein RBM8A (Y14) and MAGOH localize to centrosome in human A549 cells. Histochem Cell Biol2013; 141: 101–9.
22.
AlbersCAPaulDSSchulzeHFresonKStephensJCSmethurstPAJolleyJDCvejicAKostadimaMBertonePBreuningMHDebiliNDeloukasPFavierRFiedlerJHobbsCMHuangNHurlesMEKiddleGKrapelsINurdenPRuivenkampCALSambrookJGSmithKStempleDLStraussGThysCvan GeetCNewbury-EcobROuwehandWHGhevaertC. Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome. Nat Genet2012; 44: 435–9.
23.
HsulaWHsuMLiCChuangTWLinRITarnWY. Phosphorylation of Y14 modulates its interaction with proteins involved in mRNA metabolism and influences its methylation. J Biol Chem2005; 280: 34507–12.
24.
KinoshitaEKinoshita-KikutaEKoikeT. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc2009; 4: 1513–21.
25.
KinoshitaEKinoshita-KikutaETakiyamaKKoikeT. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics2006; 5: 749–57.
26.
MaruSIshigakiYShinoharaNTakataTTomosugiNNonomuraK. Inhibition of mTORC2 but not mTORC1 up-regulates E-cadherin expression and inhibits cell motility by blocking HIF-2α expression in human renal cell carcinoma. J Urol2013; 189: 1921–9.
27.
ZhaoXNogawaAMatsunagaTTakegamiTNakagawaHIshigakiY. Proteasome inhibitors and knockdown of SMG1 cause accumulation of Upf1 and Upf2 in human cells. Int J Oncol2014; 44: 222–8.
Bettencourt-DiasMGloverDM. Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol2007; 8: 451–63.
30.
MaHTPoonRY. How protein kinases co-ordinate mitosis in animal cells. Biochem J2011; 435: 17–31.
31.
Kinoshita-KikutaEAokiYKinoshitaEKoikeT. Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis. Mol Cell Proteomics2007; 6: 356–66.
32.
HosokawaTSaitoTAsadaAFukunagaKHisanagaS. Quantitative measurement of in vivo phosphorylation states of Cdk5 activator p35 by Phos-tag SDS-PAGE. Mol Cell Proteomics2010; 9: 1133–43.
