Regulation of FXR1 by alternative splicing is required for muscle development and controls liquid-like condensates in muscle cells

Abstract

Fragile-X mental retardation autosomal homolog-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and missplicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine and arginine-rich intrinsicallydisordered domain; these domains are known to promote biomolecular condensation.Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNAdependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease. KEYWORDS FXR1, alternative splicing, biomolecular condensation, myogenesis, Xenopus, muscle HIGHLIGHTS • The muscle-specific exon 15 impacts FXR1 functions • Alternative splicing of FXR1 is tissue-and developmental stage specific • FXR1 forms RNA-dependent condensates • Splicing regulation changes FXR1 condensate properties

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

“…We also fused a nuclear export signal (NES) to APEX to serve as a cytoplasmic control bait (Hung et al, 2016), which was used previously in SG APEX studies (Markmiller et al, 2018). To avoid overexpression artifacts, including aberrations of organelle assembly dynamics or size (Anderson and Kedersha, 2008), tet-inducible FMR1-APEX and FXR1-APEX constructs were transfected to CRISPR-edited U2OS cells lacking FMR1/FXR1/FXR2 (Smith et al, 2019), whereas a tet-inducible G3BP1-APEX construct was transfected to a previously described U2OS line lacking G3BP1/G3BP2 (Kedersha et al, 2016). We titrated induction of APEX expression by tetracycline, in isolated clones, to approximate endogenous expression levels and to be comparable across all baits (Figures S1A and S1B).…”

Spatio-temporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

“…We also fused a nuclear export signal (NES) to APEX to serve as a cytoplasm reference. To avoid overexpression artefacts including aberrations of organelle assembly dynamics or size , tet-inducible FMR1-APEX and FXR1-APEX constructs were transfected to CRISPR-edited U2OS cells lacking FMR1/FXR1/FXR2 (Smith et al, 2019), whereas a tet-inducible G3BP1-APEX construct was transfected to a previously described U2OS line lacking G3BP1/G3BP2 (Kedersha et al, 2016). We selected single clones that displayed comparable construct expression and titrated induction by tetracycline to approximate endogenous expression levels and to be comparable across all baits ( Fig.…”