Developing an E. coli model for investigating FET protein phase separation

Abstract

Some proteins in cells have the ability to bind RNA, rather than DNA, to regulate transcription in a manner comparable to traditional and DNA‐binding transcription factors. One such protein is FUS (FUsed in Sarcoma). FUS has prominent roles in the neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as sarcoma development. Nevertheless, the basic mechanism for FUS acting in normal biology, and disease, is unknown. The challenge in figuring out this mechanism arises because of the intrinsic properties of FUS as a mostly disordered protein that readily phase separates as part of its cellular functions. We have developed an E. coli model to investigate phase separation and granule formation for recombinantly expressed human FUS protein in the natural, highly crowded environment universally found in cells. We employ chemical crosslinking and chromatographic separation to observe FUS transitioning from a monomer to phase separated state. This E. coli model can provide a robust platform for large and parallel evaluation of protein phase separation, by offering rapid cell and protein production, sub‐cellular separation into small complexes and granules, and classification for structure/function relationships involving the low complexity amino acid sequences that are responsible for these unusual biophysical properties observed in FUS.Support or Funding InformationNational Institutes of Health, American Cancer Society, Alfred P. Sloan Foundation