ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Akira Ishiguro,Jun Lu,Daisaku Ozawa,Yoshitaka Nagai,Akira Ishihama

doi:10.1016/j.jbc.2021.101284

Akira Ishiguro, Jun Lu + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101284

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Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid–liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.

Highlights

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) are characterized by the progressive degeneration of nerve cells in the brain and spinal cord [1, 2]
Some of the mRNAs that are transported to the distal areas for local translation have more specific functions than general localization signals [57]
Our observations revealed that fused in sarcoma (FUS) forms specific complexes with target RNAs and exhibits transformation through liquid–liquid phase separation (LLPS) and liquid-to-solid transition (LST) in G4 structure-dependent manner

Summary

Introduction

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) are characterized by the progressive degeneration of nerve cells in the brain and spinal cord [1, 2]. Even though the characterization of mutants and the analysis of proteinaceous inclusions have provided important clues for elucidation how these gene products are connected with the diseases, the molecular mechanisms of ALS and FTLD remain unclear Along this line, one important issue is that these two proteins bind to mRNA containing G-quadruplex (G4) and transport to distal neurites for local translation [7, 8]. The purpose of this study is to clarify how FUS-G4-RNA interactions contribute to LLPS and/or LST and whether ALS-linked FUS mutations affect their phase behavior For this purpose, we constructed an in vitro system for observation of the formation and phase transition of FUS RNP condensates using the purified full-length wild-type and mutant FUS proteins and a set of FUS-binding target RNAs. Results described clearly indicated that FUS forms specific complexes with target RNAs in G4 structure-dependent manner and exhibits transformation through LLPS and LST pathways. Multiple modules of FUS protein are involved

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