MSP58/MCRS1是一種核仁蛋白，過去被報導參與調控細胞增生、細胞轉形與老化的相關訊息傳遞途徑。在文獻中指出MSP58的蛋白質異形體MCRS2胺基酸序列上包含了N端一段核定位訊號 (nuclear localization signal, NLS)與C端一段核仁定位訊號 (nucleolar localization signal, NoLS)。我們利用酵母菌雙雜合篩選 (yeast two-hybrid screening)發現MSP58蛋白與importin α1蛋白之間有交互作用，因此在我們的研究中想試圖確認MSP58的NLS與NoLS序列。利用點突變技術 (site-specific mutagenesis)與免疫螢光染色分析 (immunofluorescence analysis)，我們發現了在MSP58胺基酸序列上有兩段NLS序列，分別在胺基酸32到56 (NLS1)與117到123 (NLS2) 位置，並且確認在這些序列上進行突變時會使MSP58蛋白無法進入細胞核中。更進一步發現在NLS1上包含調控蛋白質進入核仁的NoLS序列。接著我們研究發現在HT1080細胞中，MSP58序列上的核定位訊號NLS與核仁定位訊號NoLS對於抑制細胞增生與活化p53/p21訊息傳遞途徑扮演相當重要的角色。並且透過qRT-PCR與luciferase reporter assay實驗我們證實了MSP58會調控核醣體基因的轉錄活性。最後我們透過酵母菌雙雜合篩選發現了許多會與MSP58蛋白結合的核仁蛋白，並且驗證在細胞中MSP58蛋白會與這些核仁蛋白形成蛋白質複合體，進而參與調控核醣體基因的轉錄作用。

The 58-kDa microspherule protein (MSP58; MCRS1) is a nucleolar protein controlling the cell proliferation, transformation and senescence, It had been found that there is a typical nuclear localization signal (NLS) and a bipartite nucleolar localization signal (NoLS) within MCRS2. Additionally, using the yeast two-hybrid system, we identified importin α1 as a MSP58-interacting protein. Therefore we tried to determine the presence of NLS and NoLS within the MSP58. Using site-specific mutagenesis and immunofluorescence analysis, we have identified two NLSs sequence from amino acids 32 to 56 (NLS1) and 117 to 123 (NLS2), and shows that mutating these sequences eliminates the ability of the protein to enter the nucleus. Moreover, NLS1 comprise motifs that are required for nucleolar localization. We further demonstrated that nuclear/nucleolar localization of MSP58 are
required for suppression of cell growth and activation of p53/p21 pathway in HT1080 cells. Analysis by qRT-PCR and luciferase reporter assay revealed that expression of MSP58 regulated transcriptional activity of the rDNA promoter in a context-dependent manner. Finally, we identified several nucleolar proteins as novel MSP58-interacting proteins by yeast two-hybrid screen. These results indicate that MSP58 complexes are involved in the regulation of ribosomal gene transcription in cells.

1. Ren, Y., Busch, R. K., Perlaky, L., and Busch, H. (1998) The 58-kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120. Eur J Biochem 253, 734-742
2. Davidovic, L., Bechara, E., Gravel, M., Jaglin, X. H., Tremblay, S., Sik, A., Bardoni, B., and Khandjian, E. W. (2006) The nuclear microspherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons. Hum Mol Genet 15, 1525-1538
3. Lin, D. Y., and Shih, H. M. (2002) Essential role of the 58-kDa microspherule protein in the modulation of Daxx-dependent transcriptional repression as revealed by nucleolar sequestration. J Biol Chem 277, 25446-25456
4. Ivanova, A. V., Ivanov, S. V., and Lerman, M. L. (2005) Association, mutual stabilization, and transcriptional activity of the STRA13 and MSP58 proteins. Cell Mol Life Sci 62, 471-484
5. Shimono, K., Shimono, Y., Shimokata, K., Ishiguro, N., and Takahashi, M. (2005) Microspherule protein 1, Mi-2beta, and RET finger protein associate in the nucleolus and up-regulate ribosomal gene transcription. J Biol Chem 280, 39436-39447
6. Wu, J. L., Lin, Y. S., Yang, C. C., Lin, Y. J., Wu, S. F., Lin, Y. T., and Huang, C. F. (2009) MCRS2 represses the transactivation activities of Nrf1. BMC Cell Biol 10, 9
7. Bader, A. G., Schneider, M. L., Bister, K., and Hartl, M. (2001) TOJ3, a target of the v-Jun transcription factor, encodes a protein with transforming activity related to human microspherule protein 1 (MCRS1). Oncogene 20, 7524-7535
8. Karagiannidis, A. I., Bader, A. G., Hartl, M., and Bister, K. (2008) TOJ3, a v-jun target with intrinsic oncogenic potential, is directly regulated by Jun via a novel AP-1 binding motif. Virology 378, 371-376
9. Song, H., Li, Y., Chen, G., Xing, Z., Zhao, J., Yokoyama, K. K., Li, T., and Zhao, M. (2004) Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length. Biochem Biophys Res Commun 316, 1116-1123
10. Hsu, C. C., Lee, Y. C., Yeh, S. H., Chen, C. H., Wu, C. C., Wang, T. Y., Chen, Y. N., Hung, L. Y., Liu, Y. W., Chen, H. K., Hsiao, Y. T., Wang, W. S., Tsou, J. H., Tsou, Y. H., Wu, M. H., Chang, W. C., and Lin, D. Y. (2012) 58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway. J Biol Chem 287, 22533-22548
11. Shi, H., Li, S. J., Zhang, B., Liu, H. L., and Chen, C. S. (2012) Expression of MSP58 in human colorectal cancer and its correlation with prognosis. Med Oncol 29, 3136-3142
12. Zhong, M., Zhang, X., Li, B., Chen, C. S., Ji, G. L., Li, S. X., Bi, D. Q., Zhao, Q. C., and Shi, H. (2013) Expression of MSP58 in hepatocellular carcinoma. Med Oncol 30, 539
13. Xu, C. S., Zheng, J. Y., Zhang, H. L., Zhao, H. D., Zhang, J., Wu, G. Q., Wu, L., Wang, Q., Wang, W. Z., and Zhang, J. (2012) MSP58 knockdown inhibits the proliferation of esophageal squamous cell carcinoma in vitro and in vivo. Asian Pac J Cancer Prev 13, 3233-3238
14. Lin, W., Zhang, J., Zhang, J., Liu, X., Fei, Z., Li, X., Davidovic, L., Tang, Z., Shen, L., Deng, Y., Yang, A., Han, H., Zhang, X., and Yao, L. (2009) RNAi-mediated inhibition of MSP58 decreases tumour growth, migration and invasion in a human glioma cell line. J Cell Mol Med 13, 4608-4622
15. Andersen, D. S., Raja, S. J., Colombani, J., Shaw, R. L., Langton, P. F., Akhtar, A., and Tapon, N. (2010) Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription. Mol Cell Biol 30, 4744-4755
16. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) A short amino acid sequence able to specify nuclear location. Cell 39, 499-509
17. Corbett, A. H., and Silver, P. A. (1997) Nucleocytoplasmic transport of macromolecules. Microbiol Mol Biol Rev 61, 193-211
18. Nakielny, S., and Dreyfuss, G. (1999) Transport of proteins and RNAs in and out of the nucleus. Cell 99, 677-690
19. Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E., and Goldberg, M. W. (2000) The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. J Cell Sci 113 ( Pt 10), 1651-1659
20. Faustino, R. S., Nelson, T. J., Terzic, A., and Perez-Terzic, C. (2007) Nuclear transport: target for therapy. Clin Pharmacol Ther 81, 880-886
21. Raska, I., Koberna, K., Malinsky, J., Fidlerova, H., and Masata, M. (2004) The nucleolus and transcription of ribosomal genes. Biol Cell 96, 579-594
22. Pelletier, G., Stefanovsky, V. Y., Faubladier, M., Hirschler-Laszkiewicz, I., Savard, J., Rothblum, L. I., Cote, J., and Moss, T. (2000) Competitive recruitment of CBP and Rb-HDAC regulates UBF acetylation and ribosomal transcription. Mol Cell 6, 1059-1066
23. Zhai, W., and Comai, L. (2000) Repression of RNA polymerase I transcription by the tumor suppressor p53. Mol Cell Biol 20, 5930-5938
24. Arabi, A., Wu, S., Ridderstrale, K., Bierhoff, H., Shiue, C., Fatyol, K., Fahlen, S., Hydbring, P., Soderberg, O., Grummt, I., Larsson, L. G., and Wright, A. P. (2005) c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat Cell Biol 7, 303-310
25. Grandori, C., Gomez-Roman, N., Felton-Edkins, Z. A., Ngouenet, C., Galloway, D. A., Eisenman, R. N., and White, R. J. (2005) c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat Cell Biol 7, 311-318
26. Xiao, J., Liu, C. C., Chen, P. L., and Lee, W. H. (2001) RINT-1, a novel Rad50-interacting protein, participates in radiation-induced G(2)/M checkpoint control. J Biol Chem 276, 6105-6111
27. Hirose, H., Arasaki, K., Dohmae, N., Takio, K., Hatsuzawa, K., Nagahama, M., Tani, K., Yamamoto, A., Tohyama, M., and Tagaya, M. (2004) Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi. Embo j 23, 1267-1278
28. Arasaki, K., Taniguchi, M., Tani, K., and Tagaya, M. (2006) RINT-1 regulates the localization and entry of ZW10 to the syntaxin 18 complex. Mol Biol Cell 17, 2780-2788
29. Aoki, T., Ichimura, S., Itoh, A., Kuramoto, M., Shinkawa, T., Isobe, T., and Tagaya, M. (2009) Identification of the neuroblastoma-amplified gene product as a component of the syntaxin 18 complex implicated in Golgi-to-endoplasmic reticulum retrograde transport. Mol Biol Cell 20, 2639-2649
30. Kong, L. J., Meloni, A. R., and Nevins, J. R. (2006) The Rb-related p130 protein controls telomere lengthening through an interaction with a Rad50-interacting protein, RINT-1. Mol Cell 22, 63-71
31. Lin, X., Liu, C. C., Gao, Q., Zhang, X., Wu, G., and Lee, W. H. (2007) RINT-1 serves as a tumor suppressor and maintains Golgi dynamics and centrosome integrity for cell survival. Mol Cell Biol 27, 4905-4916
32. Kuo, C. W., Wang, W. H., and Liu, S. T. (2011) Mapping signals that are important for nuclear and nucleolar localization in MCRS2. Mol Cells 31, 547-552
33. Shi, H., Chen, S., Jin, H., Xu, C., Dong, G., Zhao, Q., Wang, W., Zhang, H., Lin, W., Zhang, J., Davidovic, L., Yao, L., and Fan, D. (2009) Downregulation of MSP58 inhibits growth of human colorectal cancer cells via regulation of the cyclin D1-cyclin-dependent kinase 4-p21 pathway. Cancer Sci 100, 1585-1590
34. Wu, L., Zhang, Z. G., Qin, H. Z., Zhang, J., Gao, G. D., Lin, W., Wang, J., and Zhang, J. (2012) Downregulation of MSP58 suppresses cell proliferation in neuroblastoma cell lines. Neuroreport 23, 932-936
35. Arasaki, K., Takagi, D., Furuno, A., Sohda, M., Misumi, Y., Wakana, Y., Inoue, H., and Tagaya, M. (2013) A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex. Mol Biol Cell 24, 2907-2917
36. Arasaki, K., Uemura, T., Tani, K., and Tagaya, M. (2007) Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin. Biochem Biophys Res Commun 359, 811-816
37. Sun, Y., Shestakova, A., Hunt, L., Sehgal, S., Lupashin, V., and Storrie, B. (2007) Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis. Mol Biol Cell 18, 4129-4142