Local translation in nuclear condensate amyloid bodies

Abstract

Biomolecular condensates concentrate molecules to facilitate basic biochemical processes, including transcription and DNA replication. While liquid-like condensates have been ascribed various functions, solid-like condensates are generally thought of as amorphous sites of protein storage. Here, we show that solid-like amyloid bodies coordinate local nuclear protein synthesis (LNPS) during stress. On stimulus, translationally active ribosomes accumulate along fiber-like assemblies that characterize amyloid bodies. Mass spectrometry analysis identified regulatory ribosomal proteins and translation factors that relocalize from the cytoplasm to amyloid bodies to sustain LNPS. These amyloidogenic compartments are enriched in newly transcribed messenger RNA by Heat Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal intergenic spacer noncoding RNA (rIGSRNA) that constructs amyloid bodies prevents recruitment of the nuclear protein synthesis machinery, abolishes LNPS, and impairs the nuclear HSF1 response. We propose that amyloid bodies support local nuclear translation during stress and that solid-like condensates can facilitate complex biochemical reactions as their liquid counterparts can.

Keywords: HSR; Hsp70; acidosis; hypoxia; long noncoding RNA.

Fig. 1.

Physiological stressors activate local nuclear protein synthesis. (A) Puromycin immunofluorescence in intact acidotic or thermal-stressed cells; 100:1 anisomycin competition abolishes signal. (B) In vitro puromycylation schematic. Cells are permeabilized on coverslips with 0.1% Nonidet P-40 and washed to complete cytosolic removal before incubation with 6′FAM-puromycin or puromycin at 4 °C. (C) FAM-puromycin signal in permeabilized thermal-stressed cells lacking cytosol; 100:1 anisomycin competition abolishes signal. Signal is observed in entire cellular population of thermal-stressed cells. (D) Competition for A-site–binding assays with anisomycin eliminate FAM-puromycin signal in thermal-stressed and acidotic cells. (E) Western blot analysis of in vitro puromycylation in cytosolic (1:5 diluted) and nuclear fractions. Bio-Rad precision plus protein ladder included. (F) Complementation assay shows no accumulation of soluble puromycylated proteins in heat-shocked cells. (Scale bars: 5 μm.) Pixel intensity values depicted are ×10⁴. Error bars represent SEM. *P < 0.05.

Fig. 2.

LNPS in solid condensate amyloid bodies. (A) Superresolution microscopy of puromycin. (B) FAM-puromycin LNPS signal colocalizes with Amylo-glo, a fluorescent amyloid-specific histochemical tracer. (C) rIGSRNA depletion abolishes FAM-puromycin LNPS signal. (D) Effect of rIGSRNA depletion on FAM-puromycin LNPS signal. (E) Effect of rIGSRNA depletion on cytoplasmic and LNPS puromycin signal in thermal-stressed cells. (F) LNPS puromycin signal correlates with percentage of cells containing amyloid bodies. Measurements were taken for up to 2 h of heat shock followed by a total of 6 h in recovery. (Scale bars: 5 μm.) Pixel intensity values depicted are ×10⁴. Error bars represent SEM. *P < 0.05.

Fig. 3.

Stress-induced relocalization of key regulatory ribosomal proteins to amyloid bodies. (A) Selected Rps identified by MS-SILAC analysis of nucleoli versus amyloid bodies. Raw ratios are depicted. One-fold enrichment signifies no change between basal and stress treatment. (B) Superresolution microscopy of Rps26. (C) Western blot analysis of Rp localization. CDC73: amyloid-body marker. (D) PLA of Rps26 to puromycin reveals a quantifiable loss of proximity between Rps26 and puromycin antibodies in nuclei of thermal-stressed cells. (E) rIGSRNA depletion impairs accumulation of Rpl24 in amyloid bodies. (F) Pretreatment with 2 μg/mL Harringtonine (Harr) or 100 μM aurintricarboxylic acid (ATA) abolishes FAM-puromycin LNPS signal in thermal-stressed cells, (G) quantified by ImageJ. (Scale bars: 5 μm.) Pixel intensity values depicted are ×10⁴. Error bars represent SEM. *P < 0.05.

Fig. 4.

The amyloid-body protein synthesis machinery. (A) Immunofluorescence (IF) of eIF4H and eIF4A1. (B) PLA of eIF4H to puromycin reveals a quantifiable (C) loss of proximity between eIF4H and puromycin antibodies in nuclei of heat-shocked cells. (D) rIGSRNA depletion in heat shock impairs accumulation of eIF4H in amyloid bodies. (E) rIGSRNA depletion impairs PLA signal of eIF4H and puromycin in thermal-stressed cells in a (F) quantifiable manner. (G) In vitro FAM-puromycin signal in thermal-stressed eIF4H-depleted versus eIF4A1- or eIF5A1-depleted cells, (H) quantified by ImageJ. (I) IF of Met-tRNA-synthetase. (Scale bars: 5 μm.) Pixel intensity values depicted are ×10⁴. Error bars represent SEM. *P < 0.05.

Fig. 5.

HSF1 activation drives LNPS. (A) LNPS in cells pretreated with 100 μM DRB for 15 min. (B) LNPS signal in siRNA-mediated HSF1-depleted thermal-stressed cells, (C) quantified by ImageJ. (D) Gene ontology (GO) enrichment analysis of RNA sequencing performed on purified amyloid bodies. P values are included for each GO term. (E) List of qPCR-validated mature mRNAs enriched in amyloid bodies from thermal-stressed cells. (F) RNA-FISH of Hsp70 mRNA signal, sensitive to KRIBB11 and RNase treatment. (G) RNA-FISH of Hsp70 mRNA following rIGSRNA depletion. (H) IF of endogenous Hsp70 in rIGSRNA-depleted thermal-stressed cells, (I) quantified by ImageJ. (J) IF of eIF4H in HSF1-depleted thermal-stressed. (K) HSF1 depletion impairs PLA signal of Rps26 and puromycin in thermal-stressed cells. (Scale bars: 5 μm.) Pixel intensity values depicted are ×10⁴. Error bars represent SEM. *P < 0.05.