The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation

Laura R Ganser; Amirhossein Ghanbari Niaki; Xincheng Yuan; Ethan Huang; Dahlia Deng; Nathalie A Djaja; Yingda Ge; Alanna Craig; Olivia Langlois; Sua Myong

doi:10.1016/j.str.2023.11.006

The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation

Structure. 2024 Feb 1;32(2):177-187.e5. doi: 10.1016/j.str.2023.11.006. Epub 2023 Dec 8.

Authors

Affiliations

¹ Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
² Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA; Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA; Department of Biology, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA.
³ Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA; Department of Biology, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA.
⁴ Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA; Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA; Department of Biology, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, USA. Electronic address: smyong@jhu.edu.

Abstract

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Humans
Phase Separation*
RNA* / chemistry
RNA-Binding Motifs*
RNA-Binding Protein FUS* / chemistry

Substances

RNA
FUS protein, human
RNA-Binding Protein FUS

Abstract

Publication types

MeSH terms

Substances

Grants and funding