AJMB  Vol.3 No.1 , January 2013
Characterization of the role of Smu1 in nuclear localization of splicing factors in the mammalian temperature-sensitive mutant
Abstract
A temperature-sensitive (ts) mutant of the CHO-K1 cell line, tsTM18, grows at 340C but not at 390C. Smu1 is the gene responsible for ts defects of tsTM18 cells. Previously, we found that the Smu1 ts defect altered the localization (as indicated by enlargement of speckles) of SRSF1 (SF2/ASF) in tsTM18 cells cultured at 390C, suggesting a functional association between Smu1 and SRSF1. Speckles are subnuclear structures that may function as storage/assembly/ modification compartments to supply splicing factors to active transcription sites. The effect of the ts defect of Smu1 on the localization of other factors related to splicing has not been characterized yet. The mechanisms underlying the enlargement of speckles of SRSF1 remain unclear. In the present study, we found that the ts defect of Smu1 affected the nuclear localization of a splicing factor, SRSF2 (SC35), and factors involved in the exon-exon junction complex, Y14 and ALY. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the ts defect of Smu1 affected alternative splicing of endogenous Clk1/ Sty and SRSF2 genes. Mammalian Clk family kinases are shown to phosphorylate serine/arginine (SR) proteins in vitro and SRSF1 in vivo. RT-PCR analysis of Clk1/Sty showed an accumulation of the truncated form lacking kinase activity in tsTM18 cells incubated at 39?C. These data indicate that an accumulation of kinase-negative Clk1/Sty may lead to alteration of the localization of factors related to splicing resulting in the enlargement of speckles.

Cite this paper
Sugaya, K. , Ishihara, Y. , Sugaya, K. and Inoue, S. (2013) Characterization of the role of Smu1 in nuclear localization of splicing factors in the mammalian temperature-sensitive mutant. American Journal of Molecular Biology, 3, 38-44. doi: 10.4236/ajmb.2013.31005.
References

[1]   Fu, X.D. (1995) The superfamily of arginine/serine-rich splicing factors. RNA, 1, 663-680.

[2]   Manley, J.L. and Tacke, R. (1996) SR proteins and splicing control. Genes and Development, 10, 1569-1579. doi:10.1101/gad.10.13.1569

[3]   Graveley, B.R. (2000) Sorting out the complexity of SR protein functions. RNA, 6, 1197-1211. doi:10.1017/S1355838200000960

[4]   Le Hir, H., Izaurralde, E., Maquat, L.E. and Moore, M.J. (2000) The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO Journal, 19, 6860-6869. doi:10.1093/emboj/19.24.6860

[5]   Le Hir, H., Moore, M.J. and Maquat, L.E. (2000) PremRNA splicing alters mRNP composition: Evidence for stable association of proteins at exon-exon junctions. Genes and Development, 14, 1098-1108. doi:10.1101/gad.14.9.1098

[6]   Lamond, A.I. and Spector, D.L. (2003) Nuclear speckles: A model for nuclear organelles. Nature Reviews Molecular Cell Biology, 4, 605-612. doi:10.1038/nrm1172

[7]   Tsuji, H., Matsudo, Y., Tsuji, S., Hanaoka, F., Hyodo, M. and Hori, T. (1990) Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature. Somatic Cell and Molecular Genetics, 16, 461-476. doi:10.1007/BF01233196

[8]   Sugaya, K., Hongo, E. and Tsuji, H. (2005) A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity. Experimental Cell Research, 306, 242-251. doi:10.1016/j.yexcr.2005.02.017

[9]   Sugaya, K., Ishihara, Y. and Sugaya, K. (2011) Enlargement of speckles of SF2/ASF due to loss of function of Smu1 is characterized in the mammalian temperature-sensitive mutant. RNA Biology, 8, 488-495. doi:10.4161/rna.8.3.14656

[10]   Ge, H. and Manley, J.L. (1990) A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell, 62, 25-34. doi:10.1016/0092-8674(90)90236-8

[11]   Krainer, A.R., Conway, G.C. and Kozak, D. (1990) The essential pre-mRNA splicing factor SF2 influences 5’ splice site selection by activating proximal sites. Cell, 62, 35-42. doi:10.1016/0092-8674(90)90237-9

[12]   Li, X. and Manley, J.L. (2005) Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell, 122, 365-378. doi:10.1016/j.cell.2005.06.008

[13]   Hongo, E., Ishihara, Y., Sugaya, K. and Sugaya, K. (2008) Characterization of cells expressing RNA polymerase II tagged with green fluorescent protein: Effect of ionizing irradiation on RNA synthesis. International Journal of Radiation Biology, 84, 778-787. doi:10.1080/09553000802345936

[14]   Sugaya, K., Hongo, E., Ishihara, Y. and Tsuji, H. (2006) The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant. Journal of Cell Science, 119, 4944-4951. doi:10.1242/jcs.03288

[15]   Fu, X.D. and Maniatis, T. (1990) Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature, 343, 437-441. doi:10.1038/343437a0

[16]   Ballut, L., Marchadier, B., Baguet, A., Tomasetto, C., Seraphin, B. and Le Hir, H. (2005) The exon junction core complex is locked onto RNA by inhibition of eIF4-AIII ATPase activity. Nature Structural & Molecular Biology, 12, 861-869. doi:10.1038/nsmb990

[17]   Tange, T.O., Shibuya, T., Jurica, M.S. and Moore, M.J. (2005) Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core. RNA, 11, 1869-1883. doi:10.1261/rna.2155905

[18]   Le Hir, H. and Andersen, G.R. (2008) Structural insights into the exon junction complex. Current Opinion in Structural Biology, 18, 112-119. doi:10.1016/j.sbi.2007.11.002

[19]   Duncan, P.I., Stojdl, D.F., Marius, R.M. and Bell, J.C. (1997) In Vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Molecular and Cellular Biology, 17, 5996-6001.

[20]   Colwill, K., Pawson, T., Andrews, B., Prasad, J., Manley, J.L., Bell, J.C. and Duncan, P.I. (1996) The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO Journal, 15, 265-275.

[21]   Nayler, O., Stamm, S. and Ullrich, A. (1997) Characterization and comparison of four serineand arginine-rich (SR) protein kinases. Biochemical Journal, 326, 693-700.

[22]   Caceres, J.F., Screaton, G.R. and Krainer, A.R. (1998) A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes and Development, 12, 55-66. doi:10.1101/gad.12.1.55

[23]   Lai, M.C., Lin, R.I. and Tarn, W.Y. (2003) Differential effects of hyperphosphorylation on splicing factor SRp55. Biochemical Journal, 371, 937-945. doi:10.1042/BJ20021827

[24]   Pilch, B., Allemand, E., Facompre, M., Bailly, C., Riou, J.F., Soret, J. and Tazi, J. (2001) Specific Inhibition of Serine-and Arginine-rich Splicing Factors Phosphorylation, Spliceosome Assembly, and Splicing by the Antitumor Drug NB-506. Cancer Research, 61, 6876-6884.

[25]   Soret, J., Gabut, M., Dupon, C., Kohlhagen, G., Stevenin, J., Pommier, Y. and Tazi, J. (2003) Altered Serine/Arginine-Rich Protein Phosphorylation and Exonic Enhancer-Dependent Splicing in Mammalian Cells Lacking To-poisomerase I. Cancer Research, 63, 8203-8211.

[26]   Sureau, A., Gattoni, R., Dooghe, Y., Stevenin, J. and Soret, J. (2001) SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs. EMBO Journal, 20, 1785-1796. doi:10.1093/emboj/20.7.1785

[27]   Schmidt, U., Im, K.B., Benzing, C., Janjetovic, S., Rippe, K., Lichter, P. and Wachsmuth, M. (2009) Assembly and mobility of exon-exon junction complexes in living cells. RNA, 15, 862-876. doi:10.1261/rna.1387009

[28]   Xiao, S.H. and Manley, J.L. (1997) Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes and Development, 11, 334-344. doi:10.1101/gad.11.3.334

[29]   Caceres, J.F., Misteli, T., Screaton, G.R., Spector, D.L. and Krainer, A.R. (1997) Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. Journal of Cell Biology, 138, 225-238. doi:10.1083/jcb.138.2.225

[30]   Misteli, T., Cáceres, J.F., Clement, J.Q., Krainer, A.R., Wilkinson, M.F. and Spector, D.L. (1998) Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. Journal of Cell Biology, 143, 297-307. doi:10.1083/jcb.143.2.297

[31]   Lai, M.C., Lin, R.I., Huang, S.Y., Tsai, C.W. and Tarn, W.Y. (2000) A human importin-β family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins. Journal of Biological Chemistry, 275, 7950-7957. doi:10.1074/jbc.275.11.7950

[32]   Lai, M.C., Lin, R.I. and Tarn, W.Y. (2001) Transportin-SR2 mediates nuclear import of phosphorylated SR proteins. Proceedings of the National Academy of Sciences of the United States of America, 98, 10154-10159. doi:10.1073/pnas.181354098

[33]   Lin, S., Xiao, R., Sun, P., Xu, X. and Fu, X.D. (2005) Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation. Molecular Cell, 20, 413-425. doi:10.1016/j.molcel.2005.09.015

 
 
Top