Journal of the Korea Academia-Industrial cooperation Society (한국산학기술학회논문지)

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
권호 표지
DOI QR코드

DOI QR Code

Analysis of the Role of RGG box of human hnRNP A1 protein

인간 hnRNP A1 단백질에 포함된 RGG 상자의 기능 분석

  • Choi, Mieyoung (Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University)
  • 최미영 (선문대학교 BT융합 제약공학과)
  • Received : 2017.11.03
  • Accepted : 2017.12.08
  • Published : 2017.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

This study analyzed the effects of RGG box of hnRNP A1 on its subcellular localization and stabilization of hnRNP A1 over a three year period from October 2014. First, a 6R/K mutation in RGG box was generated, and pcDNA1-HA-hnRNP A1(6R/K) was constructed. The subcellular localization of hnRNP A1(6R/K) from the HeLa cells transfected with this plasmid DNA was analyzed by immunofluorescence microscopy. HA-hnRNP A1(6R/K) was found to exhibit nuclear and cytoplasmic fluorescence. The stability of hnRNP A1(6R/K) was checked by Western blot analysis using the expressed protein from the HeLa cells transfected with the pcDNA1-HA-hnRNP A1(6R/K). The results show that HA-hnRNP A1(6R/K) has a smaller size. These confirm that HA-hnRNP A1(6R/K) is localized both in the nuclear and cytoplasm, not because 6R/K mutation affects the nuclear localization of hnRNP A1, but because 6R/K mutation causes hnRNP A1(6R/K) to cleave at the mutation or near the mutation site. The cleaved protein fragment, which lacks the M9 domain (i.e. nuclear localization signal of hnRNP A1), did not exhibit nuclear fluorescence. This suggests that the arginines of RGG box in hnRNP A1 play an important role in stabilizing hnRNP A1. An analysis of the RNA-binding ability of hnRNP A1(6R/K) expressed and purified from bacteria will be a subsequent research project.

본 연구는 hnRNP A1 단백질에 포함되어 있는 RGG 상자가 이 단백질의 세포내 위치에 미치는 영향 및 이 단백질의 안정화에 미치는 영향을 분석하는 것을 목적으로 하며 2014년 10월부터 약 3년 동안 수행되었다. 이를 위해 먼저, RGG상자 내의 6개의 아르기닌을 라이신으로 돌연변이를 만든 다음 pcDNA1-HA-hnRNP A1(6R/K)를 재조합하였다. 다음, 이 플라스미드 DNA를 HeLa 세포에 형질주입하여 HA-hnRNP A1(6R/K) 단백질의 세포내 위치에 미치는 영향을 면역형광현미경법으로 분석하였다. 그 결과 hnRNP A1(6R/K) 단백질은 (wt 단백질처럼 핵에 위치하지 못하고) 핵과 세포질 모두에 퍼져 있는 것을 확인하였다. hnRNP A1(6R/K)의 안정성을 조사하기 위해서는 pcDNA1-HA-hnRNP A1(6R/K)를 HeLa 세포에 형질 주입시킨 다음 발현된 단백질을 웨스턴 블랏 실험으로 분석하였는데, 그 결과 HA-hnRNP A1(6R/K)는 (잘려서) 크기가 작아진 것을 확인할 수 있었다. 이 결과들로부터 HA-hnRNP A1(6R/K)가 핵과 세포질 모두에 퍼져 있는 것은 6R/K 돌연변이가 hnRNP A1의 핵 위치 능력에 영향을 미쳤기 때문이 아니라 6R/K 돌연변이가 일어난 부위 또는 그 부근이 절단되어 hnRNP A1의 핵 위치 신호(nuclear localization singal)인 M9 도메인이 들어있는 C-말단 부위를 잃었기 때문이라는 점을 확인 할 수 있었다. 본 연구의 결과들은 hnRNP A1에 있는 RGG 상자의 아르기닌이 hnRNP A1의 안정성에 중요한 역할을 한다는 것을 제시한다. 세균에서 발현시켜서 정제된 hnRNP A1(6R/K)의 RNA-결합 능력에 대한 영향 분석은 추후 과제로 남겨둔다.

Keywords

References

  1. J. Jean-Philippe, S. Paz, M. Caputi, "hnRNP A1: The Swiss Army Knife of Gene Expression", Int. J. Mol. Sci., 14, pp. 18999-19024, 2013. DOI: https://doi.org/10.3390/ijms140918999
  2. B-J. Thiele, A. Doller, T. Kahne, R. Pregla, R. Hetzer, V. Regitz-Zagrosek, "RNA-binding proteins heterogeneous nuclear ribonucleoprotein A1, E1, and K are involved in post-transcriptional control of collagen I and III synthesis", Circ. Res., 95, pp. 1058-1066, 2004. DOI: https://doi.org/10.1161/01.RES.0000149166.33833.08
  3. F. Wang, X. Fu, P. Chen, P. Wu, X. Fan, N. Li, H. Z hu, T. T. Jia, H. Ji, Z. Wang, C. C. Wong, R. Hu, J. Hui, "SPSB1-mediated HnRNP A1 ubiquitylation regulat es alternative splicing and cell migration in EGF signaling", Cell Res., 27, pp. 540-558, 2017. DOI: https://doi.org/10.1038/cr.2017.7
  4. A. Cammas, F. Pileur, S. Bonnal, S. M. Lewis, N. L eveque, M. Holick, S. Vagner, "Cytoplasmic relocaliza tion of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs". Mol. Biol. Cell, 18, pp. 5048-5059, 2007. DOI: https://doi.org/10.1091/mbc.E07-06-0603
  5. H-J. Kim, H-R. Lee, J-Y. Seo, H. G. Ryu, K-H. Lee, D-Y. Kim, K-T. Kim, "Heterogeneous nuclear ribonucleoprotein A1 regulates rhythmic synthesis of mouse Nfil3 protein via IRES-mediated translation", Sci Rep. 7, pp. 42882-428895, 2017. DOI: https://doi.org/10.1038/srep42882
  6. M. Iijima, M. Suzuki, A. Tanabe, A. Nishimura, M. Yamada, "Two motifs essential for nuclear import of the hnRNP A1 nucleocytoplasmic shuttling sequence M9 core", FEBS Lett. 580, pp. 1365-1370, 2006. DOI: https://doi.org/10.1016/j.febslet.2006.01.058
  7. M. Kiledjian, G. Dreyfuss, "Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box", EMBO J., 11, pp. 2655-2664, 1992.
  8. J. Ye, N. Beetz, S. O'Keeffe, J. C. Tapia, L. Macpherson, W. V. Chen, R. Bassel-Duby, E. N. Olson, T. Maniatis, "hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function", Proc. Natl. Acad. Sci. U S A. 112, pp. 3020-3029, 2015. DOI: https://doi.org/10.1073/pnas.1508461112
  9. M. T. Bedford, S. G. Clarke, "Protein arginine methylation in mammals: who, what, and why", Mol. Cell, 33, pp. 1-13, 2009. DOI: https://doi.org/10.1016/j.molcel.2008.12.013
  10. S. Kim, B. M. Merrill, R. Rajpurohit, A. Kumar, K. L. Stone, V. V. Papov, J. M. Schneiders, W. Szer, S. H. Wilson, W. K. Paik, K R. Williams, "Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif", Biochemistry, 36, pp. 5185-5192, 1997. DOI: https://doi.org/10.1021/bi9625509
  11. G. Gao, S. Dhar, M. T. Bedford, "PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1", Nucleic Acids Res., 45, pp. 4359-4369, 2017. DOI: https://doi.org/10.1093/nar/gkw1367
  12. S-Y. Lee. S. K. Jang, M. Choi, "Human hnRNP L shuttles between nucleus and cytoplasm", Korean J. Genetics, 25, pp. 141-145, 2003.
  13. B. R. Glick, J. J. Pasternak, C. L. Patten, Molecular biotechnology: principles and applications of recombinant DNA. 4th ed., p. 292-295, ASM press, 2010.
  14. T. Takahashi, T. Nakamura, A. Hayashi, M. Kamei, M. Nakabayashi, A. A. Okada, N. TomitaN, Y. Kaneda, Y. Tano, "Inhibition of experimental choroidal neovascularization by overexpression of tissue inhibitor of metalloproteinases-3 in retinal pigment epithelium cells", Am. J. Ophthalmol., 130, pp. 774-781, 2000. DOI: https://doi.org/10.1016/S0002-9394(00)00772-8
  15. L. R. Friend, S. P. Han, J. A. Rothnagel, R. Smith, "Differential subnuclear localisation of hnRNPs A/B is dependent on transcription and cell cycle stage", Biochem. Biophys. Acta, 1783, pp. 1972-1980, 2008. DOI: https://doi.org/10.1016/j.bbamcr.2008.05.021
  16. D. 1. Boquet, C. Creminon, G. Clement, Y. Frobert, M. C. Nevers, S. Essono, J. Grassi, "Quantitative Measurement of Bitagged Recombinant Proteins Using an Immunometric Assay: Application to an Anti-Substance P Recombinant Antibody", Anal Biochem., 284, pp. 221-230, 2000. DOI: https://doi.org/10.1006/abio.2000.4711
  17. H. Seo, I. Lee, H. S. Chung, G-U. Bae, M. Chang, E. Song, M. J. Kim, "ATP5B regulates mitochodrial fission and fusion in mammalian cells", Animal Cells and Systems, 20, pp. 157-164, 2016. https://doi.org/10.1080/19768354.2016.1188855