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Phase Separation of Purified Human LSM4 Protein

  • STRUCTURAL-FUNCTIONAL ANALYSIS OF BIOPOLYMERS AND THEIR COMPLEXES
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Abstract

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. The results suggest that full-length human LSM4 protein can undergo liquid–liquid phase separation in vitro.

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Funding

This work was supported by the National Natural Science Foundation of China for Youth (Grant no. 81602275).

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Authors and Affiliations

  1. Health Care Office, Service Bureau of The General Administration of Affairs, The Central Military Commission, 100034, Bei**g, China

    H. Li & W. W. Liu

  2. Department of Obstetrics and Gynecology, Seventh Medical Center of Chinese People’s Liberation Army General Hospital, 100071, Bei**g, China

    Y. Ju

  3. Department of Minimally Invasive Gynecologic Center, Bei**g Obstetrics and Gynecology Hospital, Capital Medical University, Bei**g Maternal and Child Health Care Hospital, 100006, Bei**g, China

    Y. Y. Ma, H. Ye & N. Li

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Contributions

NL wrote the manuscript and designed the study. HY analyzed and interpreted the results of the LSM4 cloning study. HL and YM prepared the protein purification and FPLC. HL and WL designed and performed phase separation experiments. YJ performed the PONDR analysis. All authors read and approved the final manuscript. NL and HL confirmed the authenticity of all raw data. HL and YJ contributed equally.

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Correspondence to H. Ye or N. Li.

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COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no competing interests. The article contains no studies in which animals were used.

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Li, H., Ju, Y., Liu, W.W. et al. Phase Separation of Purified Human LSM4 Protein. Mol Biol 57, 127–135 (2023). https://doi.org/10.1134/S0026893323010065

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  • DOI: https://doi.org/10.1134/S0026893323010065

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