Ribonucleoprotein (RNP) granules are membraneless organelles, consisting of high local concentrations of RNA and proteins bearing intrinsically disordered regions (IDRs). They are formed by liquid-liquid phase separation (LLPS). In neurodegenerative diseases such as ALS, mutations in granule proteins such as FUS and TDP-43 accelerate abnormal liquid to solid transition of RNP granules, leading to formation of fiber-like structures. Methods to study granules must be carefully selected based on the stage of granule’s life. Here we describe a strategic combination of single-molecule biophysical and ensemble biochemical techniques that may be employed to extract insightful information about early stages of RNP granule formation. Protein-RNA interaction and stoichiometry of the complex in the early soluble stage of RNP assembly can be probed by single-molecule FRET (smFRET) assay and electrophoretic mobility shift assay (EMSA), respectively. RNP-RNP interaction that likely contributes to RNP nucleation can be reported on by a smFRET-based RNA annealing assay. The next stage in the assembly pathway, that is, phase separation from diffused to liquid-like droplets, may be monitored by a phase separation assay. Finally, RNP granules isolated from mammalian cells can be investigated using a unique single-molecule pull-down (SiMPull) assay.
CITATION STYLE
Sarkar, J., & Myong, S. (2018). Single-molecule and ensemble methods to probe initial stages of RNP granule assembly. In Methods in Molecular Biology (Vol. 1814, pp. 325–338). Humana Press Inc. https://doi.org/10.1007/978-1-4939-8591-3_19
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