Phase Separation of Purified Human LSM4 Protein

Capa

Citar

Texto integral

Acesso aberto Acesso aberto
Acesso é fechado Acesso está concedido
Acesso é fechado Somente assinantes

Resumo

Liquid–liquid phase separation of proteins occur in a number of biological processes, such as regulation of transcription, processing, and RNA maturation. Sm-like protein 4 (LSM4) is involved in multiple processes, including pre-mRNA splicing and P-bodies assembly. Before investigating the involvement of LSM4 in the separation of the two liquid phases during RNA processing or maturation, the separation of the liquid phases in an in vitro preparation of LSM4 protein should be first be detected. The mCherry-LSM4 plasmid was derived from pET30a and used to isolate mCherry-LSM4 protein from prokaryotic cells (Escherichia coli strain BL21). The mCherry-LSM4 protein was purified using Ni-NTA resin. The protein was further purified by fast protein liquid chromatography. Delta-Vision wide-field fluorescence microscopy was used to observe the dynamic liquid–liquid phase separation of the LSM4 protein in vitro. Analysis of the LSM4 protein structure using the Predictor of Natural Disordered Regions database revealed that its C-terminus contains a low complexity domain. A purified preparation of full-length human LSM4 protein was obtained from E. coli. Human LSM4 was shown to provide concentration-dependent separation of liquid–liquid phases in vitro in buffer with crowding reagents. Salts in high concentration and 1,6-hexanediol block the LSM4-induced separation of the two liquid phases. In addition, in vitro fusion of LSM4 protein droplets is observed. These results indicate that the full-length human LSM4 protein has the ability to form liquid inclusions and induce liquid–liquid phase separation in vitro.

Sobre autores

H. Li

Health Care Office, Service Bureau of The General Administration of Affairs, The Central Military Commission

Email: linaog@126.com
China, 100034, Beijing

Y. Ju

Department of Obstetrics and Gynecology, The Seventh Medical Center of Chinese People’s Liberation Army General Hospital

Email: linaog@126.com
China, 100071, Beijing

W. Liu

Health Care Office, Service Bureau of The General Administration of Affairs, The Central Military Commission

Email: linaog@126.com
China, 100034, Beijing

Y. Ma

Department of Minimally Invasive Gynecologic Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital

Email: linaog@126.com
China, 100006, Beijing

H. Ye

Department of Minimally Invasive Gynecologic Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital

Autor responsável pela correspondência
Email: yehong@ccmu.edu.cn
China, 100006, Beijing

N. Li

Department of Minimally Invasive Gynecologic Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital

Autor responsável pela correspondência
Email: linaog@126.com
China, 100006, Beijing

Bibliografia

  1. Banani S.F., Lee H.O., Hyman A.A., Rosen M.K. (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell. Biol. 18(5), 285‒298.
  2. Mitrea D.M., Cika J.A., Stanley C.B., Nourse A., Onuchic P.L., Banerjee P.R., Phillips A.H., Park C.G., Deniz A.A., Kriwacki R.W. (2018) Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation. Nat. Commun. 9(1), 842.
  3. McSwiggen D.T., Hansen A.S., Teves S.S., Marie-Nelly H., Hao Y., Heckert A.B., Umemoto K.K., Dugast-Darzacq C., Tjian R., Darzacq X. (2019) Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation. Elife. 8, e47098.
  4. Hyman A.A., Weber C.A., Julicher F. (2014) Liquid-liquid phase separation in biology. Annu. Rev. Cell. Dev. Biol. 30, 39‒58.
  5. Aguzzi A., Altmeyer M. (2016) Phase separation: linking cellular compartmentalization to disease. Trends Cell Biol. 26(7), 547‒558.
  6. Bergeron-Sandoval L.P., Safaee N., Michnick S.W. (2016) Mechanisms and consequences of macromolecular phase separation. Cell. 165(5), 1067‒1079.
  7. Toretsky J.A., Wright P.E. (2014) Assemblages: functional units formed by cellular phase separation. J. Cell Biol. 206(5), 579‒588.
  8. Lyon A.S., Peeples W.B., Rosen M.K. (2021) A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 22(3), 215‒235.
  9. Sanulli S., Trnka M.J., Dharmarajan V., Tibble R.W., Pascal B.D., Burlingame A.L., Griffin P.R., Gross J.D., Narlikar G.J. (2019) HP1 reshapes nucleosome core to promote phase separation of heterochromatin. Nature. 575(7782), 390‒394.
  10. Zhao Y.G., Zhang H. (2020) Phase separation in membrane biology: the interplay between membrane-bound organelles and membraneless condensates. Dev. Cell. 55(1), 30‒44.
  11. Jiang H, Wang S., Huang Y., He X., Cui H., Zhu X, Zheng Y. (2015) Phase transition of spindle-associated protein regulate spindle apparatus assembly. Cell. 163(1), 108‒122.
  12. Huang Y., Li T., Ems-McClung S.C., Walczak C.E., Prigent C., Zhu X., Zhang X., Zheng Y. (2018) Aurora A activation in mitosis promoted by BuGZ. J. Cell. Biol. 217(1), 107‒116.
  13. Lee K.H., Zhang P., Kim H.J., Mitrea D.M., Sarkar M., Freibaum B.D., Cika J., Coughlin M., Messing J., Molliex A., Maxwell B.A., Kim N.C., Temirov J., Moore J., Kolaitis R.M., Shaw T.I., Bai B., Peng J., Kriwacki R.W., Taylor J.P. (2016) C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell. 167(3), 774‒788. e717.
  14. Altmeyer M., Neelsen K.J., Teloni F., Pozdnyakova I., Pellegrino S., Grøfte M., Rask M.D., Streicher W., Jungmichel S., Nielsen M.L., Lukas J. (2015) Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat. Commun. 6, 8088.
  15. Boeynaems S., Alberti S., Fawzi N.L., Mittag T., Polymenidou M., Rousseau F., Schymkowitz J., Shorter J., Wolozin B., Van Den Bosch L., Tompa P., Fuxreiter M. (2018) Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28(6), 420‒435.
  16. Shin Y., Brangwynne C.P. 2017) Liquid phase condensation in cell physiology and disease. Science. 357, 6357.
  17. Patel A., Lee H.O., Jawerth L., Maharana S., Jahnel M., Hein M.Y., Stoynov S., Mahamid J., Saha S., Franzmann T.M., Pozniakovski A., Poser I., Maghelli N., Royer L.A., Weigert M., Myers E.W., Grill S., Drechsel D., Hyman A.A., Alberti S. (2015) A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 162(5), 1066‒1077.
  18. Pannone B.K., Kim S.D., Noe D.A., Wolin S.L. (2001) Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein. Genetics. 158(1), 187‒196.
  19. Arribas-Layton M., Dennis J., Bennett E.J., Damgaard C.K., Lykke-Andersen J. (2016) The C-terminal RGG domain of human Lsm4 promotes processing body formation stimulated by arginine dimethylation. Mol. Cell. Biol. 36(17), 2226‒2235.
  20. Reijns M.A., Alexander R.D., Spiller M.P., Beggs J.D. (2008) A role for Q/N-rich aggregation-prone regions in P-body localization. J. Cell Sci. 121(Pt 15), 2463‒2472.
  21. Roth A.J., Shuman S., Schwer B. (2018) Defining essential elements and genetic interactions of the yeast Lsm2-8 ring and demonstration that essentiality of Lsm2-8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24. RNA. 24(6), 853‒864.
  22. Lyons S.M., Ricciardi A.S., Guo A.Y., Kambach C., Marzluff W.F. (2014) The C-terminal extension of Lsm4 interacts directly with the 3' end of the histone mRNP and is required for efficient histone mRNA degradation. RNA. 20(1), 88‒102.
  23. Decker C.J., Teixeira D., Parker R. (2007) Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell Biol. 179(3), 437‒449.
  24. Mennie A.K., Moser B.A., Nakamura T.M. (2018) LARP7-like protein Pof8 regulates telomerase assembly and poly (A)+ TERRA expression in fission yeast. Nat. Commun. 9(1), 1‒12.
  25. Adamson B.S., Smogorzewska A., Sigoillot F.D., King R.W., Elledge S.J. (2012) A genome-wide study of homologous recombination in mammalian cells identifies RBMX, a novel component of the DNA damage response. Nat. Cell Biol. 14(3), 318‒328.
  26. Alabrudzinska M., Skoneczny M., Skoneczna A. (2011) Diploid-specific genome stability genes of S. cerevisiae: genomic screen reveals haploidization as an escape from persisting DNA rearrangement stress. PLoS One. 6(6), e21124.
  27. Lin Y., Protter D.S., Rosen M.K., Parker R. (2015) Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell. 60(2), 208‒219.
  28. Linding R., Jensen L.J., Diella F., Bork P., Gibson T.J., Russell R.B. (2003) Protein disorder prediction: implications for structural proteomics. Structure. 11(11), 1453‒1459.
  29. Schuster B.S., Reed E.H., Parthasarathy R., Jahnke C.N., Caldwell R.M., Bermudez J.G., Ramage H., Good M.C., Hammer D.A. (2018) Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 9(1), 2985.
  30. Uversky V.N. (2017) Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder. Curr. Opin. Struct. Biol. 44, 18‒30.
  31. Sasahara K., McPhie P., Minton A.P. (2003) Effect of dextran on protein stability and conformation attributed to macromolecular crowding. J. Mol. Biol. 326(4), 1227‒1237.
  32. Alberti S., Saha S., Woodruff J.B., Franzmann T.M., Wang J., Hyman A.A. (2018) A user’s guide for phase separation assays with purified proteins. J. Mol. Biol. 430(23), 4806‒4820.
  33. Alberti S., Gladfelter A., Mittag T. (2019) Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell. 176(3), 419‒434.
  34. Verdone L., Galardi S., Page D., Beggs J.D. (2004) Lsm proteins promote regeneration of pre-mRNA splicing activity. Curr. Biol. 14(16), 1487‒1491.
  35. Rao B.S., Parker R. (2017) Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 114(45), E9569‒E9578.
  36. Tang W., Kannan R., Blanchette M., Baumann P. (2012) Telomerase RNA biogenesis involves sequential binding by Sm and Lsm complexes. Nature. 484(7393), 260‒264.

Declaração de direitos autorais © H. Li, Y. Ju, W.W. Liu, Y.Y. Ma, H. Ye, N. Li, 2023

Este site utiliza cookies

Ao continuar usando nosso site, você concorda com o procedimento de cookies que mantêm o site funcionando normalmente.

Informação sobre cookies