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Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

Abstract

Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

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Figure 1: Schematic illustration of iPOLYMER and in silico analysis on network formation.
Figure 2: iPOLYMER puncta formation in living cells.
Figure 3: Biophysical and ultrastructural analysis of iPOLYMER in living cells.
Figure 4: In vitro characterization of iPOLYMER.
Figure 5: Reconstituting RNA granules by using iPOLYMER as scaffold.
Figure 6: Light-inducible and reversible iPOLYMER-LI succeeded in RNA granule reconstitution.

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Acknowledgements

We are grateful to N. Kedersha and P. Anderson who provided helpful discussions and reagents related to stress granules, to J. L. Pfaltz who collaborated with A.S.A. to develop a modified C++ code for identifying chordless cycles in graphs, and to R. Reed, A. Ewald, H. Sesaki, M. Iijima and S. Regot for sharing their resources for our experiments. We also extend our appreciation to J. P. Gong, I. Hamachi, R. Yoshida for valuable comments on our work. This work was mainly supported by the Johns Hopkins University Catalyst Fund to T.I., and in part by the National Institutes of Health (NIH) (GM092930, DK102910, CA103175 and DK089502 to T.I., and T32GM007445 to A.S.), and the National Science Foundation (NSF) (CCF-1217213 to J.G.).

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H.N., A.A.L. and T.I. conceived the project. H.N., A.A.L., A.S., Y.-C.L., M.T., R.D. and D.B., performed molecular biology as well as cell biology experiments. H.N., A.A.L., S.R. and A.S. purified proteins under the guidance of W.H. and S.B.G. The biochemical and biophysical experiments were mostly performed by H.N. and A.A.L., and partially by S.R. and Y.-C.L. H.N., A.A.L. and T.I. wrote the manuscript with the help of J.G. A.S.A. and J.G. developed the computational model, analysed the computational results, and wrote the computational parts of the paper. A.S.A. wrote appropriate code and conducted the computational experiments. S.W. performed correlated EM measurement and analysis. E.R. and B.H. performed development and demonstration of light-inducible iPOLYMER with H.N.

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Correspondence to Ali Sobhi Afshar or Takanari Inoue.

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Nakamura, H., Lee, A., Afshar, A. et al. Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions. Nature Mater 17, 79–89 (2018). https://doi.org/10.1038/nmat5006

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