Abstract
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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
