NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Vyšlo v časopise:
NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly. PLoS Genet 10(2): e32767. doi:10.1371/journal.pgen.1004110
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1004110
Souhrn
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Zdroje
1. Cavdar KocE, BurkhartW, BlackburnK, MoseleyA, SpremulliLL (2001) The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present. J Biol Chem 276: 19363–19374.
2. KocEC, BurkhartW, BlackburnK, MoyerMB, SchlatzerDM, et al. (2001) The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present. J Biol Chem 276: 43958–43969.
3. SiibakT, RemmeJ (2010) Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified. RNA 16: 2023–2032.
4. MetodievMD, LeskoN, ParkCB, CamaraY, ShiY, et al. (2009) Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome. Cell Metab 9: 386–397.
5. LeeKW, Okot-KotberC, LacombJF, BogenhagenDF (2013) Mitochondrial rRNA Methyltransferase Family Members are Positioned to Modify Nascent rRNA in Foci Near the mtDNA Nucleoid. J Biol Chem 288(43): 31386–99.
6. BaerRJ, DubinDT (1981) Methylated regions of hamster mitochondrial ribosomal RNA: structural and functional correlates. Nucleic Acids Res 9: 323–337.
7. DecaturWA, FournierMJ (2002) rRNA modifications and ribosome function. Trends Biochem Sci 27: 344–351.
8. CamaraY, Asin-CayuelaJ, ParkCB, MetodievMD, ShiY, et al. (2011) MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian mitochondrial ribosome. Cell Metab 13: 527–539.
9. SpahrH, HabermannB, GustafssonCM, LarssonNG, HallbergBM (2012) Structure of the human MTERF4-NSUN4 protein complex that regulates mitochondrial ribosome biogenesis. Proc Natl Acad Sci U S A 109: 15253–15258.
10. YakubovskayaE, GujaKE, MejiaE, CastanoS, HambardjievaE, et al. (2012) Structure of the essential MTERF4:NSUN4 protein complex reveals how an MTERF protein collaborates to facilitate rRNA modification. Structure 20: 1940–1947.
11. GuXR, GustafssonC, KuJ, YuM, SantiDV (1999) Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli. Biochemistry 38: 4053–4057.
12. AndersenNM, DouthwaiteS (2006) YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407. J Mol Biol 359: 777–786.
13. PurtaE, O'ConnorM, BujnickiJM, DouthwaiteS (2008) YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962. J Mol Biol 383: 641–651.
14. DemirciH, LarsenLH, HansenT, RasmussenA, CadambiA, et al. (2010) Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus. RNA 16: 1584–1596.
15. WredenbergA, LagougeM, BraticA, MetodievMD, SpahrH, et al. (2013) MTERF3 Regulates Mitochondrial Ribosome Biogenesis in Invertebrates and Mammals. PLoS Genet 9: e1003178.
16. DagaA, MicolV, HessD, AebersoldR, AttardiG (1993) Molecular characterization of the transcription termination factor from human mitochondria. J Biol Chem 268: 8123–8130.
17. KruseB, NarasimhanN, AttardiG (1989) Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination. Cell 58: 391–397.
18. MartinM, ChoJ, CesareAJ, GriffithJD, AttardiG (2005) Termination factor-mediated DNA loop between termination and initiation sites drives mitochondrial rRNA synthesis. Cell 123: 1227–1240.
19. TerziogluM, RuzzenenteB, HarmelJ, MourierA, JemtE, et al. (2013) MTERF1 binds mtDNA to prevent transcriptional interference at the light-strand promoter but is dispensable for rRNA gene transcription regulation. Cell Metab 17: 618–626.
20. WenzT, LucaC, TorracoA, MoraesCT (2009) mTERF2 regulates oxidative phosphorylation by modulating mtDNA transcription. Cell Metab 9: 499–511.
21. ParkCB, Asin-CayuelaJ, CamaraY, ShiY, PellegriniM, et al. (2007) MTERF3 is a negative regulator of mammalian mtDNA transcription. Cell 130: 273–285.
22. RuzzenenteB, MetodievMD, WredenbergA, BraticA, ParkCB, et al. (2011) LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs. EMBO J 31: 443–456.
23. SchaeferM, PollexT, HannaK, LykoF (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res 37: e12.
24. KingMY, RedmanKL (2002) RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41: 11218–11225.
25. RedmanKL (2006) Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase. Biomacromolecules 7: 3321–3326.
26. SeibelP, Di NunnoC, KukatC, SchaferI, Del BoR, et al. (2008) Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids. Nucleic Acids Res 36: 5872–5881.
27. LafontaineD, VandenhauteJ, TollerveyD (1995) The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev 9: 2470–2481.
28. LafontaineDL, PreissT, TollerveyD (1998) Yeast 18S rRNA dimethylase Dim1p: a quality control mechanism in ribosome synthesis? Mol Cell Biol 18: 2360–2370.
29. ThammanaP, HeldWA (1974) Methylation of 16S RNA during ribosome assembly in vitro. Nature 251: 682–686.
30. XuZ, O'FarrellHC, RifeJP, CulverGM (2008) A conserved rRNA methyltransferase regulates ribosome biogenesis. Nat Struct Mol Biol 15: 534–536.
31. DemirciH, GregoryST, DahlbergAE, JoglG (2008) Crystal structure of the Thermus thermophilus 16 S rRNA methyltransferase RsmC in complex with cofactor and substrate guanosine. J Biol Chem 283: 26548–26556.
32. ConnollyK, CulverG (2009) Deconstructing ribosome construction. Trends Biochem Sci 34: 256–263.
33. ChenY, Sierzputowska-GraczH, GuentherR, EverettK, AgrisPF (1993) 5-Methylcytidine is required for cooperative binding of Mg2+ and a conformational transition at the anticodon stem-loop of yeast phenylalanine tRNA. Biochemistry 32: 10249–10253.
34. AlexandrovA, ChernyakovI, GuW, HileySL, HughesTR, et al. (2006) Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell 21: 87–96.
35. MotorinY, HelmM (2011) RNA nucleotide methylation. Wiley Interdiscip Rev RNA 2: 611–631.
36. KruegerF, AndrewsSR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27: 1571–1572.
37. LangmeadB, TrapnellC, PopM, SalzbergSL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.
38. EnriquezJA, Perez-MartosA, Lopez-PerezMJ, MontoyaJ (1996) In organello RNA synthesis system from mammalian liver and brain. Methods Enzymol 264: 50–57.
39. Fernandez-VizarraE, FerrinG, Perez-MartosA, Fernandez-SilvaP, ZevianiM, et al. (2010) Isolation of mitochondria for biogenetical studies: An update. Mitochondrion 10: 253–262.
40. CoteC, PoirierJ, BouletD (1989) Expression of the mammalian mitochondrial genome. Stability of mitochondrial translation products as a function of membrane potential. J Biol Chem 264: 8487–8490.
41. SieversF, WilmA, DineenD, GibsonTJ, KarplusK, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539.
42. NicholasKB, H.BN, Deerfield IIDW (1997) GeneDoc: Analysis and Visualization of Genetic Variation. embnetnews 4: 4.
43. LarssonNG, WangJ, WilhelmssonH, OldforsA, RustinP, et al. (1998) Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet 18: 231–236.
44. UleJ, JensenK, MeleA, DarnellRB (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37: 376–386.
45. HafnerM, LandthalerM, BurgerL, KhorshidM, HausserJ, et al. (2010) PAR-CliP–a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp (41): pii: 2034.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
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