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. 2001 Nov;7(11):1531–1542. doi: 10.1017/s135583820101442x

Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein.

H Brahms 1, L Meheus 1, V de Brabandere 1, U Fischer 1, R Lührmann 1
PMCID: PMC1370196  PMID: 11720283

Abstract

Arginine residues in RG-rich proteins are frequently dimethylated posttranslationally by protein arginine methyltransferases (PRMTs). The most common methylation pattern is asymmetrical dimethylation, a modification important for protein shuttling and signal transduction. Symmetrically dimethylated arginines (sDMA) have until now been confined to the myelin basic protein MBP and the Sm proteins D1 and D3. We show here by mass spectrometry and protein sequencing that also the human Sm protein B/B' and, for the first time, one of the Sm-like proteins, LSm4, contain sDMA in vivo. The symmetrical dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to the Tudor domain of the "survival of motor neurons" protein (SMN): inhibition of dimethylation by S-adenosylhomocysteine (SAH) abolished the binding of D1, D3, B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine dipeptides, but not asymmetrically modified or nonmodified peptides, specifically inhibited the interaction of D1, D3, B/B', LSm4, and UsnRNPs with SMN-Tudor. Recombinant D1 and a synthetic peptide could be methylated in vitro by both HeLa cytosolic S100 extract and nuclear extract; however, only the cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. Our demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in tri-UsnRNP biogenesis and mRNA decay. Our findings also have interesting implications for the understanding of the aetiology of spinal muscular atrophy (SMA).

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Selected References

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  1. Achsel T., Brahms H., Kastner B., Bachi A., Wilm M., Lührmann R. A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro. EMBO J. 1999 Oct 15;18(20):5789–5802. doi: 10.1093/emboj/18.20.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Achsel T., Stark H., Lührmann R. The Sm domain is an ancient RNA-binding motif with oligo(U) specificity. Proc Natl Acad Sci U S A. 2001 Mar 20;98(7):3685–3689. doi: 10.1073/pnas.071033998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldwin G. S., Carnegie P. R. Specific enzymic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin. Science. 1971 Feb 12;171(3971):579–581. doi: 10.1126/science.171.3971.579. [DOI] [PubMed] [Google Scholar]
  4. Bouveret E., Rigaut G., Shevchenko A., Wilm M., Séraphin B. A Sm-like protein complex that participates in mRNA degradation. EMBO J. 2000 Apr 3;19(7):1661–1671. doi: 10.1093/emboj/19.7.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brahms H., Raker V. A., van Venrooij W. J., Lührmann R. A major, novel systemic lupus erythematosus autoantibody class recognizes the E, F, and G Sm snRNP proteins as an E-F-G complex but not in their denatured states. Arthritis Rheum. 1997 Apr;40(4):672–682. doi: 10.1002/art.1780400412. [DOI] [PubMed] [Google Scholar]
  6. Brahms H., Raymackers J., Union A., de Keyser F., Meheus L., Lührmann R. The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies. J Biol Chem. 2000 Jun 2;275(22):17122–17129. doi: 10.1074/jbc.M000300200. [DOI] [PubMed] [Google Scholar]
  7. Branscombe T. L., Frankel A., Lee J. H., Cook J. R., Yang Z., Pestka S., Clarke S. PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins. J Biol Chem. 2001 Jun 18;276(35):32971–32976. doi: 10.1074/jbc.M105412200. [DOI] [PubMed] [Google Scholar]
  8. Bühler D., Raker V., Lührmann R., Fischer U. Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. Hum Mol Genet. 1999 Dec;8(13):2351–2357. doi: 10.1093/hmg/8.13.2351. [DOI] [PubMed] [Google Scholar]
  9. Camasses A., Bragado-Nilsson E., Martin R., Séraphin B., Bordonné R. Interactions within the yeast Sm core complex: from proteins to amino acids. Mol Cell Biol. 1998 Apr;18(4):1956–1966. doi: 10.1128/mcb.18.4.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Charroux B., Pellizzoni L., Perkinson R. A., Yong J., Shevchenko A., Mann M., Dreyfuss G. Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli. J Cell Biol. 2000 Mar 20;148(6):1177–1186. doi: 10.1083/jcb.148.6.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper M., Johnston L. H., Beggs J. D. Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins. EMBO J. 1995 May 1;14(9):2066–2075. doi: 10.1002/j.1460-2075.1995.tb07198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer U., Liu Q., Dreyfuss G. The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell. 1997 Sep 19;90(6):1023–1029. doi: 10.1016/s0092-8674(00)80368-2. [DOI] [PubMed] [Google Scholar]
  14. Fischer U., Lührmann R. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science. 1990 Aug 17;249(4970):786–790. doi: 10.1126/science.2143847. [DOI] [PubMed] [Google Scholar]
  15. Fischer U., Sumpter V., Sekine M., Satoh T., Lührmann R. Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap. EMBO J. 1993 Feb;12(2):573–583. doi: 10.1002/j.1460-2075.1993.tb05689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friesen W. J., Dreyfuss G. Specific sequences of the Sm and Sm-like (Lsm) proteins mediate their interaction with the spinal muscular atrophy disease gene product (SMN). J Biol Chem. 2000 Aug 25;275(34):26370–26375. doi: 10.1074/jbc.M003299200. [DOI] [PubMed] [Google Scholar]
  17. Friesen W. J., Massenet S., Paushkin S., Wyce A., Dreyfuss G. SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol Cell. 2001 May;7(5):1111–1117. doi: 10.1016/s1097-2765(01)00244-1. [DOI] [PubMed] [Google Scholar]
  18. Gary J. D., Clarke S. RNA and protein interactions modulated by protein arginine methylation. Prog Nucleic Acid Res Mol Biol. 1998;61:65–131. doi: 10.1016/s0079-6603(08)60825-9. [DOI] [PubMed] [Google Scholar]
  19. Ghosh S. K., Paik W. K., Kim S. Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme. J Biol Chem. 1988 Dec 15;263(35):19024–19033. [PubMed] [Google Scholar]
  20. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  21. He W., Parker R. Functions of Lsm proteins in mRNA degradation and splicing. Curr Opin Cell Biol. 2000 Jun;12(3):346–350. doi: 10.1016/s0955-0674(00)00098-3. [DOI] [PubMed] [Google Scholar]
  22. Hermann H., Fabrizio P., Raker V. A., Foulaki K., Hornig H., Brahms H., Lührmann R. snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions. EMBO J. 1995 May 1;14(9):2076–2088. doi: 10.1002/j.1460-2075.1995.tb07199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kambach C., Walke S., Young R., Avis J. M., de la Fortelle E., Raker V. A., Lührmann R., Li J., Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell. 1999 Feb 5;96(3):375–387. doi: 10.1016/s0092-8674(00)80550-4. [DOI] [PubMed] [Google Scholar]
  24. Krapivinsky G., Pu W., Wickman K., Krapivinsky L., Clapham D. E. pICln binds to a mammalian homolog of a yeast protein involved in regulation of cell morphology. J Biol Chem. 1998 May 1;273(18):10811–10814. doi: 10.1074/jbc.273.18.10811. [DOI] [PubMed] [Google Scholar]
  25. Lefebvre S., Burlet P., Liu Q., Bertrandy S., Clermont O., Munnich A., Dreyfuss G., Melki J. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet. 1997 Jul;16(3):265–269. doi: 10.1038/ng0797-265. [DOI] [PubMed] [Google Scholar]
  26. Lehmeier T., Foulaki K., Lührmann R. Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5. Nucleic Acids Res. 1990 Nov 25;18(22):6475–6484. doi: 10.1093/nar/18.22.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lehmeier T., Raker V., Hermann H., Lührmann R. cDNA cloning of the Sm proteins D2 and D3 from human small nuclear ribonucleoproteins: evidence for a direct D1-D2 interaction. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12317–12321. doi: 10.1073/pnas.91.25.12317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lischwe M. A., Cook R. G., Ahn Y. S., Yeoman L. C., Busch H. Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23. Biochemistry. 1985 Oct 22;24(22):6025–6028. doi: 10.1021/bi00343a001. [DOI] [PubMed] [Google Scholar]
  29. Liu Q., Fischer U., Wang F., Dreyfuss G. The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell. 1997 Sep 19;90(6):1013–1021. doi: 10.1016/s0092-8674(00)80367-0. [DOI] [PubMed] [Google Scholar]
  30. Mattaj I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. doi: 10.1016/0092-8674(86)90072-3. [DOI] [PubMed] [Google Scholar]
  31. Mayes A. E., Verdone L., Legrain P., Beggs J. D. Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J. 1999 Aug 2;18(15):4321–4331. doi: 10.1093/emboj/18.15.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meister G., Bühler D., Laggerbauer B., Zobawa M., Lottspeich F., Fischer U. Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins. Hum Mol Genet. 2000 Aug 12;9(13):1977–1986. doi: 10.1093/hmg/9.13.1977. [DOI] [PubMed] [Google Scholar]
  33. Mowen K. A., Tang J., Zhu W., Schurter B. T., Shuai K., Herschman H. R., David M. Arginine methylation of STAT1 modulates IFNalpha/beta-induced transcription. Cell. 2001 Mar 9;104(5):731–741. doi: 10.1016/s0092-8674(01)00269-0. [DOI] [PubMed] [Google Scholar]
  34. Mura C., Cascio D., Sawaya M. R., Eisenberg D. S. The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core. Proc Natl Acad Sci U S A. 2001 May 1;98(10):5532–5537. doi: 10.1073/pnas.091102298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Najbauer J., Aswad D. W. Diversity of methyl acceptor proteins in rat pheochromocytoma (PC12) cells revealed after treatment with adenosine dialdehyde. J Biol Chem. 1990 Jul 25;265(21):12717–12721. [PubMed] [Google Scholar]
  36. Nelissen R. L., Will C. L., van Venrooij W. J., Lührmann R. The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins. EMBO J. 1994 Sep 1;13(17):4113–4125. doi: 10.1002/j.1460-2075.1994.tb06729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Padgett R. A., Mount S. M., Steitz J. A., Sharp P. A. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell. 1983 Nov;35(1):101–107. doi: 10.1016/0092-8674(83)90212-x. [DOI] [PubMed] [Google Scholar]
  38. Pellizzoni L., Charroux B., Dreyfuss G. SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11167–11172. doi: 10.1073/pnas.96.20.11167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pellizzoni L., Kataoka N., Charroux B., Dreyfuss G. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell. 1998 Nov 25;95(5):615–624. doi: 10.1016/s0092-8674(00)81632-3. [DOI] [PubMed] [Google Scholar]
  40. Plessel G., Fischer U., Lührmann R. m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain. Mol Cell Biol. 1994 Jun;14(6):4160–4172. doi: 10.1128/mcb.14.6.4160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pollack B. P., Kotenko S. V., He W., Izotova L. S., Barnoski B. L., Pestka S. The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity. J Biol Chem. 1999 Oct 29;274(44):31531–31542. doi: 10.1074/jbc.274.44.31531. [DOI] [PubMed] [Google Scholar]
  42. Pu W. T., Krapivinsky G. B., Krapivinsky L., Clapham D. E. pICln inhibits snRNP biogenesis by binding core spliceosomal proteins. Mol Cell Biol. 1999 Jun;19(6):4113–4120. doi: 10.1128/mcb.19.6.4113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Raker V. A., Plessel G., Lührmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J. 1996 May 1;15(9):2256–2269. [PMC free article] [PubMed] [Google Scholar]
  44. Rawal N., Rajpurohit R., Lischwe M. A., Williams K. R., Paik W. K., Kim S. Structural specificity of substrate for S-adenosylmethionine:protein arginine N-methyltransferases. Biochim Biophys Acta. 1995 Apr 5;1248(1):11–18. doi: 10.1016/0167-4838(94)00213-z. [DOI] [PubMed] [Google Scholar]
  45. Rho J., Choi S., Seong Y. R., Cho W. K., Kim S. H., Im D. S. Prmt5, which forms distinct homo-oligomers, is a member of the protein-arginine methyltransferase family. J Biol Chem. 2001 Jan 10;276(14):11393–11401. doi: 10.1074/jbc.M008660200. [DOI] [PubMed] [Google Scholar]
  46. Salgado-Garrido J., Bragado-Nilsson E., Kandels-Lewis S., Séraphin B. Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin. EMBO J. 1999 Jun 15;18(12):3451–3462. doi: 10.1093/emboj/18.12.3451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Seipelt R. L., Zheng B., Asuru A., Rymond B. C. U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway. Nucleic Acids Res. 1999 Jan 15;27(2):587–595. doi: 10.1093/nar/27.2.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Selenko P., Sprangers R., Stier G., Bühler D., Fischer U., Sattler M. SMN tudor domain structure and its interaction with the Sm proteins. Nat Struct Biol. 2001 Jan;8(1):27–31. doi: 10.1038/83014. [DOI] [PubMed] [Google Scholar]
  49. Sumpter V., Kahrs A., Fischer U., Kornstädt U., Lührmann R. In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA. Mol Biol Rep. 1992 Sep;16(4):229–240. doi: 10.1007/BF00419662. [DOI] [PubMed] [Google Scholar]
  50. Ségault V., Will C. L., Sproat B. S., Lührmann R. In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs. EMBO J. 1995 Aug 15;14(16):4010–4021. doi: 10.1002/j.1460-2075.1995.tb00072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Séraphin B. Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs. EMBO J. 1995 May 1;14(9):2089–2098. doi: 10.1002/j.1460-2075.1995.tb07200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tharun S., He W., Mayes A. E., Lennertz P., Beggs J. D., Parker R. Yeast Sm-like proteins function in mRNA decapping and decay. Nature. 2000 Mar 30;404(6777):515–518. doi: 10.1038/35006676. [DOI] [PubMed] [Google Scholar]
  53. Uetz P., Giot L., Cagney G., Mansfield T. A., Judson R. S., Knight J. R., Lockshon D., Narayan V., Srinivasan M., Pochart P. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000 Feb 10;403(6770):623–627. doi: 10.1038/35001009. [DOI] [PubMed] [Google Scholar]
  54. Vidal V. P., Verdone L., Mayes A. E., Beggs J. D. Characterization of U6 snRNA-protein interactions. RNA. 1999 Nov;5(11):1470–1481. doi: 10.1017/s1355838299991355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Will C. L., Lührmann R. Spliceosomal UsnRNP biogenesis, structure and function. Curr Opin Cell Biol. 2001 Jun;13(3):290–301. doi: 10.1016/s0955-0674(00)00211-8. [DOI] [PubMed] [Google Scholar]
  56. Zhang D., Abovich N., Rosbash M. A biochemical function for the Sm complex. Mol Cell. 2001 Feb;7(2):319–329. doi: 10.1016/s1097-2765(01)00180-0. [DOI] [PubMed] [Google Scholar]
  57. Zhang D., Rosbash M. Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex. Genes Dev. 1999 Mar 1;13(5):581–592. doi: 10.1101/gad.13.5.581. [DOI] [PMC free article] [PubMed] [Google Scholar]

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