FUS interacts with nuclear matrix-associated protein SAFB1 as well as Matrin3 to regulate splicing and ligand-mediated transcription.

Atsushi Yamaguchi,Keisuke Takanashi

doi:10.1038/srep35195

Abstract

FUS (Fused-in-Sarcoma) is a multifunctional DNA/RNA binding protein linked to familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Since FUS is localized mainly in the nucleus with nucleo-cytoplasmic shuttling, it is critical to understand physiological functions in the nucleus to clarify pathogenesis. Here we report a yeast two-hybrid screening identified FUS interaction with nuclear matrix-associated protein SAFB1 (scaffold attachment factor B1). FUS and SAFB1, abundant in chromatin-bound fraction, interact in a DNA-dependent manner. N-terminal SAP domain of SAFB1, a DNA-binding motif, was required for its localization to chromatin-bound fraction and splicing regulation. In addition, depletion of SAFB1 reduced FUS’s localization to chromatin-bound fraction and splicing activity, suggesting SAFB1 could tether FUS to chromatin compartment thorough N-terminal DNA-binding motif. FUS and SAFB1 also interact with Androgen Receptor (AR) regulating ligand-dependent transcription. Moreover, FUS interacts with another nuclear matrix-associated protein Matrin3, which is muted in a subset of familial ALS cases and reportedly interacts with TDP-43. Interestingly, ectopic ALS-linked FUS mutant sequestered endogenous Matrin3 and SAFB1 in the cytoplasmic aggregates. These findings indicate SAFB1 could be a FUS’s functional platform in chromatin compartment to regulate RNA splicing and ligand-dependent transcription and shed light on the etiological significance of nuclear matrix-associated proteins in ALS pathogenesis.

Highlights

Chromatin is thought to be organized in ordered structures consisting of radial looped domains fixed to the nuclear matrix/scaffold, which is a proteinaceous fibrogranular framework resistant to high salt and detergent extraction
Scaffold Attachment Factor B 1 (SAFB1) protein was present in the immunoprecipitate with anti-FUS antibody, indicating the interaction between FUS and SAFB1 in sheared chromatins
Since previous report showed that Matrin[3] interacts with SAFB1 and SAFB223, we addressed whether FUS interacts with Matrin[3] in Co-IP assay

Summary

Introduction

Chromatin is thought to be organized in ordered structures consisting of radial looped domains fixed to the nuclear matrix/scaffold, which is a proteinaceous fibrogranular framework resistant to high salt and detergent extraction. Nuclear matrix-associated proteins (nuclear matrix proteins), bounding to the internal nuclear matrix/scaffold, are thought to form a skeletal nuclear framework with roles in chromatin organization, DNA replication, DNA repair, transcriptional regulation, and RNA metabolism. HEK293 with siRNA-mediated SAFB knockdown were processed for chromatin fraction assay. Attachment factor B1 (SAFB1), considered as a nuclear matrix-associated protein, was originally identified by independent two groups, based on its ability to bind to S/MAR15 or the promoter of hsp2716. Matrin[3], considered as a component of nuclear matrix-associated complex, is a DNA/RNA binding protein with multi-domains, containing NLS and NES (nuclear export signal), two zinc finger domains and two RRM, which can anchor chromosomes to the nucleus matrix thorough binding to the S/MAR. We examined the functional significance of the interplay between FUS and SAFB1 in mammalian cultured cells

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Conclusion