Local translation in nuclear condensate amyloid bodies

Significance

The central dogma of eukaryotic biology is clear: DNA replication and RNA transcription occur in the nucleus while protein synthesis takes place in the cytoplasm. While this fundamental tenet of biology is entrenched in textbooks, it has undergone recurring challenges over the decades, most notably the existence of nuclear translation. We report that solid-like condensate amyloid bodies are hubs of stress-resistant local nuclear translation in cells engaging in hypoxic fermentation or responding to high temperature. Conceptually, this paper provides a physiological context for nuclear protein synthesis while highlighting a role for solid-like condensates in coordinating complex biochemical reactions.

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.

Cells are remarkable in their ability to sustain viability under adverse environmental conditions (1). Hypoxia-induced extracellular acidosis is a common stimulus observed in an array of physiological and pathological settings including development, cancer, and ischemic episodes (2, 3). Cells acclimatize to the acidotic state by suppressing global protein synthesis among other pathways (4–6) while activating the heat shock response (HSR) involved in protein homeostasis (7–9). The master regulator of the HSR is heat shock factor 1 (HSF1) that transcribes genes encoding a wide array of chaperones, including the canonical heat shock protein 70 (Hsp70) (10). Activation of the HSR through HSF1 is believed to sustain cellular viability during periods of intense stress, such as extracellular acidosis and heat shock (9).

In addition to activating the HSR, extracellular acidosis and heat shock induce the formation of several membraneless bodies (e.g., stress granules) that have been implicated in a wide range of cellular functions, such as storage of cytoplasmic messenger RNAs (mRNAs) and nuclear splicing factors. These organelles have been described as liquid-like biomolecular condensates and are formed by a phenomenon called “phase separation” (11–14). Liquid-like biomolecular condensates are typically dynamic as their constituent proteins are highly mobile and can exchange between the phase-separated bodies and the extracellular milieu (15–18). Fermentation-induced acidosis and heat shock also stimulate the formation of amyloid bodies: nuclear organelles that can be readily distinguished from other compartments by their large electron-dense fibers that stain with various amyloidophylic dyes (19). Amyloid bodies are constructed by a class of inducible long noncoding RNA (lncRNA) derived from stimuli-specific loci of the ribosomal intergenic spacer (rIGSRNA) (20–22). Mass spectrometry and photobleaching analyses revealed that amyloid bodies are enriched in a heterogeneous population of full-length proteins, many of which are immobile (23–26). As such, amyloid bodies have been described as nondynamic, or solid-like, condensates with amyloidogenic properties (27–30) similar to Balbiani bodies in Xenopus oocytes (31). In contrast to liquid-like condensates that concentrate molecules to facilitate biochemical reactions, amyloid bodies and Balbiani bodies are believed to store proteins and induce cellular dormancy (19, 27, 31). Since amyloid bodies also comprise an array of mobile proteins (e.g., ribosomal proteins) (23), it remains unknown if these nuclear condensates have other cellular functions in addition to protein retention and metabolic depression.

Here, we report that nuclear amyloid bodies are sites of stress-resistant local nuclear protein synthesis (LNPS). Mass spectrometry with stable isotope labeling by amino acids in cell culture (MS-SILAC) cytoplasmic ribosomal proteins and translation factors that relocalize to amyloid bodies in cells responding to anaerobic fermentation or heat shock. In vitro puromycylation and proximity ligase assays uncovered ribosomal acceptor sites organized along fiber-like assemblies that characterize these solid-like condensates. Amyloid bodies anchor HSF1 target mRNAs that recruit the LNPS machinery to sustain nuclear protein synthesis. We discuss how amyloid-body protein synthesis can be compared to local translation machineries that synthesize proteins at specific cellular coordinates (32–40) during periods of inhibition of nuclear/cytoplasmic trafficking. These results identify amyloid bodies as nuclear, solid-like condensates that sustain stress-induced local translation.

Results

Physiological Stressors Induce LNPS in Amyloid Bodies.

Cells were incubated in low-oxygen tension to induce the natural acidification of their extracellular milieu as a consequence of lactic fermentation, recapitulating various in vivo conditions including ischemia (41) and microenvironments of aggressive tumors (42). Under such anaerobic acidosis conditions (37 °C, 1% O2, pH 6.0), cells remain viable while displaying a marked decrease in global translation compared to their basal (37 °C, 21% O2, pH 7.4) or anaerobic neutral (37°, 1% O2, pH 7.4) counterparts (SI Appendix, Fig. S1 A and B). To corroborate these results, we performed immunofluorescence analysis on cells incubated with puromycin (SI Appendix, Fig. S1C). Cells were treated with emetine to prevent the synthesis of new proteins and fixed with the denaturant methanol to enable the detection of free and residual translating, ribosome-associated, puromycylated peptides (43, 44). Under these experimental conditions, we observed abundant puromycin signal in the cytoplasm of cells maintained at basal (37 °C, 21% O2, pH 7.4) or hypoxic neutral (37°, 1% O2, pH 7.4) conditions (Fig. 1A). Surprisingly, puromycin signal was detected in nuclear foci, in addition to the cytoplasm, in cells engaging in anaerobic fermentation (Fig. 1A) or exposed to heat shock (43 °C, 21% O2, pH 7.4; Fig. 1A and SI Appendix, Fig. S1 D–F), another potent inhibitor of global protein synthesis (SI Appendix, Fig. S1 A–E). Previous studies revealed that a small fraction of puromycylated peptides remain bound to translating ribosomes (45, 46); this fraction may be detectable by immunofluorescence especially if concentrated in clearly defined foci. To test the possibility that elongating ribosomes form in the nucleus of cells responding to stressors, we designed an in vitro puromycylation assay adapted from well-established protocols (47–51). Cells grown on coverslips were subjected to cytoplasmic membrane solubilization followed by extensive washing to remove cytosolic components as monitored by the loss of calreticulin staining (Fig. 1B). Permeabilized cells were incubated with 6′carboxyfluorescein (6′FAM)-labeled puromycin (52) to directly monitor the puromycylation reaction without the need for immunofluorescence. The assay was done at 4 °C, which does not prevent the puromycylation reaction but considerably decreases the release of puromycylated peptides from translating ribosomes (51, 53, 54). Consistent with previous reports (47), a slightly over background nuclear 6′FAM-puromycin signal was observed in permeabilized cells that were maintained at basal conditions (Fig. 1C: first panel and fourth panel for low magnification). In sharp contrast, intense nuclear 6′FAM-puromycin signal was detected in acidotic or thermal-stressed cells (Fig. 1C: second and third panels and fifth panel for low magnification; Fig. 1D). Anisomycin competes with puromycin for ribosomal A-site binding although these molecules are structurally different and thus unlikely to bind to the same nonspecific sites (47, 55, 56). Addition of anisomycin abolished in vitro 6′FAM-puromycin incorporation (Fig. 1 C and D and SI Appendix, Fig. S1 F and G), demonstrating that the puromycylation reactions occur at bona fide ribosomal A-sites in the nucleus. Likewise, addition of anisomycin abolished puromycin signal in intact cells (Fig. 1A). The presence of nuclear ribosomes actively synthesizing proteins was further demonstrated by the detection of elongating puromycylated peptides of different lengths following Western analysis of the in vitro assay (Fig. 1 B and E). The presence of different puromycylated species confirmed that the nuclear signal is not due to contaminating cytosol, aborted small peptides, or puromycylated transfer RNAs (tRNAs). We estimate that LNPS represents roughly 5% of total protein synthesis intensity in stressed cells as assessed by Western blot analysis and cellular puromycin signal (SI Appendix, Fig. S1H). We performed a complementation assay to eliminate the unlikely possibility that in vitro nuclear puromycylation signals arise from any other contaminating cytosolic components during permeabilization. Soluble and insoluble fractions of cells maintained at 37 °C or 43 °C incubated with 6′FAM-puromycin were directly added to permeabilized cells in the presence of anisomycin (SI Appendix, Fig. S1I). We did not observe nuclear 6′FAM-puromycin following these complementation assays, demonstrating that nuclear 6′FAM-puromycin did not originate from residual, contaminating cytosolic 6′FAM-puromycylated peptides or translating ribosomes that could have entered the nucleus during permeabilization (Fig. 1F and SI Appendix, Fig. S1J). Nuclear puromycin signal was rapidly observed under stress (SI Appendix, Fig. S1D) and was completely lost within a few hours following stimuli termination (SI Appendix, Fig. S1K).

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.

Fermentation-induced extracellular acidosis and thermal stress trigger the formation of various membrane-less liquid- and solid-like compartments. High-resolution microscopy of nuclear 6′FAM-puromycin fluorescence and nuclear puromycin signal detected by immunofluorescence revealed translating ribosomes organized along fiber-like assemblies that characterize solid-like condensate amyloid bodies (Fig. 2A). The nuclear 6′FAM-puromycin and immunofluorescence signals colocalized with amyloidophylic dyes, confirming that amyloid bodies are sites of stress-induced LNPS (Fig. 2B and SI Appendix, Fig. S2A). Amyloid-body biogenesis is mediated by a class of inducible lncRNA molecules derived from stimuli-specific loci of the rDNA intergenic spacer (rIGSRNA) (SI Appendix, Fig. S2B) (19–22). Induction of these chromatin-associated lncRNA molecules remodels nucleoli into fibrous amyloid bodies (SI Appendix, Fig. S2B). Depletion of rIGS₁₆RNA and rIGS₂₂RNA, which seed amyloid bodies during heat shock, reduced nuclear 6′FAM-puromycin incorporation (Fig. 2 C and D and SI Appendix, Fig. S2C) and cellular puromycin immunofluorescence signal (SI Appendix, Fig. S2D) to levels similar to those observed in basal conditions with little effect on cytoplasmic immunofluorescence (SI Appendix, Fig. S2D). Likewise, rIGS₂₈RNA silencing, which prevents anaerobic acidosis-induced amyloid-body biogenesis, impaired LNPS under acidosis (Fig. 2 C and D and SI Appendix, Fig. S2C). These experiments eliminate any possibility that 6′FAM-puromycin nuclear signal may be caused by contaminating cytosolic ribosomes entering the nucleus during the permeabilization step as this contamination would need to be dependent on chromatin-associated lncRNA. Likewise, the loss of nuclear puromycylation signal in rIGSRNA-depleted intact cells further suggests that the aforementioned nuclear signal is not simply the consequence of diffusion of puromycylated peptides from the cytoplasm. Termination of LNPS correlated with the disassembly of amyloid bodies during stress recovery (Fig. 2F and SI Appendix, Fig. S1K). These results suggest that physiological stressors stimulate LNPS in solid-like condensate amyloid bodies.

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.

The LNPS Machinery.

Current paradigms imply that protein synthesis is strictly localized in the cytoplasm since regulatory ribosomal proteins (Rps) and translation factors reside exclusively in this subcellular domain. These models suggest that preribosomes are initially produced in nucleoli and require export to undergo the final steps of maturation (57–60) by assembling with key cytoplasmic Rps such as Rpl24 and Rpl7 (61). Interestingly, SILAC-MS analysis (19, 24) revealed that amyloid bodies not only retain, but also are broadly enriched in ribosomal proteins, including the aforementioned regulatory Rps (Fig. 3A and SI Appendix, Fig. S3A). Immunofluorescence (Fig. 3B and SI Appendix, Fig. S3 B and C) and Western blot analysis (Fig. 3C and SI Appendix, Fig. S3D) confirmed that full-length regulatory Rps accumulate in amyloid bodies along their characteristic fiber-like assemblies (Fig. 3B) in proximity to puromycylated peptides (Fig. 3D and SI Appendix, Fig. S3 E and F). Silencing of rIGSRNA prevented the recruitment of regulatory Rps to amyloid bodies (Fig. 3E and SI Appendix, Fig. S3G) as well as nuclear Rps26/puromycylated peptides proximity ligation assay (PLA) signal in cells (Fig. 3D). Based on data shown in Fig. 3 A–E that amyloid-body ribosomes harbor similar Rps to cytoplasmic ribosomes, we reasoned that LNPS should be sensitive to the same antibiotics that inhibit cytoplasmic translation. Consistent with this notion, treatment of thermal-stressed or acidotic cells with the translation initiation inhibitors harringtonine (Harr) (62) or aurintricarboxylic acid (ATA) (63) efficiently abolished 6′FAM-puromycin incorporation in the in vitro assays and nuclear puromycylation in intact cells (Fig. 3 F and G and SI Appendix, Fig. S3 H and I). Thus, amyloid bodies retain preribosomal subunits while recruiting regulatory Rps typically found in the cytoplasm to form translationally competent ribosomes.

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.

MS-SILAC also identified the enrichment in amyloid bodies of the eukaryotic translation initiation factor 4H (eIF4H) (64). Immunofluorescence analysis confirmed that eIF4H is exclusively cytoplasmic at 37 °C but relocalizes to amyloid bodies in thermal-stressed (Fig. 4A) or acidotic cells (SI Appendix, Fig. S4A), colocalizing with puromycin (SI Appendix, Fig. S4B) and amyloidophilic dyes (SI Appendix, Fig. S4A). As with Rps26, eIF4H was observed in proximity to nuclear puromycylated peptides in intact cells (Fig. 4 B and C) and in the in vitro assay (SI Appendix, Fig. S4C) which, again, excludes the possibility that nuclear PLA signal originates from diffusion of puromycylated peptides from cytoplasmic ribosomes. Depletion of rIGSRNA impaired the relocalization of eIF4H to amyloid bodies and proximity between eIF4H and nuclear puromycylated peptides, further suggesting that these solid-like condensates coordinate the assembly of the LNPS machinery (Fig. 4 D–F). Silencing of eIF4H had no discernible effect on protein synthesis intensity at 37 °C (SI Appendix, Fig. S4D) but prevented LNPS in amyloid bodies as measured by in vitro incorporation of 6′FAM-puromycin and puromycin immunofluorescence (Fig. 4 G and H and SI Appendix, Fig. S4D) without affecting the construction of these solid-like condensates (SI Appendix, Fig. S4 E and F). We suspected that additional translation factors relocalize to amyloid bodies but may have escaped detection by MS-SILAC. To test this, we performed an immunofluorescence screen of several known endogenous translation factors to examine their potential role in LNPS (SI Appendix, Fig. S4 G and H). As expected, translation factors were cytoplasmic in cells maintained at 37 °C. While many translation factors remained cytoplasmic even during stress (SI Appendix, Fig. S4G, gray), several were abundantly observed in amyloid bodies under heat shock conditions (SI Appendix, Fig. S4G, purple, black). RNA interference analysis revealed that translation factors that remained in the cytoplasm during stress did not participate in LNPS or have accessory functions (SI Appendix, Fig. S4G, blue; SI Appendix, Fig. S4 D–H). For example, efficient silencing of eIF4A1 or eIF5A1, translation initiation factors that localize to the cytoplasm at 43 °C (Fig. 4 G and H and SI Appendix, Fig. S4 F–H), had no effect on amyloid-body 6′FAM-puromycin incorporation and puromycin immunofluorescence during heat shock (Fig. 4 G and H and SI Appendix, Fig. S4D). In contrast, silencing of eIF4B that accumulates in amyloid bodies, along with eIF4H, impaired nuclear protein synthesis (SI Appendix, Fig. S4G: red, SI Appendix, Fig. S4 D and H). It has been suggested that several aminoacyl-tRNA synthetases localize to translating ribosomes, thereby marking sites of active local translation (65). Immunofluorescence analysis revealed that Met-, Lys-, and Arg-tRNA synthetases were found in amyloid bodies in a rIGSRNA-dependent manner (Fig. 4I and SI Appendix, Fig. S4 I and J), suggesting that LNPS encompasses all the major biochemical classes (i.e., ribosomes, translation factors, aminoacyl tRNA synthetases) involved in cytoplasmic protein synthesis. Put together, these results demonstrate that regulatory Rps and translation factors relocalize from the cytoplasm to amyloid bodies to participate in stress-induced local translation.

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.

HSF1 Activation Drives LNPS.

Heat shock activates a potent transcription response to coordinate the HSR. Treatment with the RNA polymerase II inhibitor 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) completely abolished 6′FAM-puromycin fluorescence and puromycin signal in amyloid bodies without affecting the construction of these solid-like condensates (Fig. 5A and SI Appendix, Fig. S5 A and B). This suggested that stress-induced de novo-transcribed mRNAs are captured by amyloid bodies for LNPS. HSF1 is a master regulator of the stress response that activates the transcription of heat shock genes. Silencing of HSF1 (Fig. 5 B and C) or treatment with an HSF1 inhibitor (SI Appendix, Fig. S5 C–E) impaired nuclear 6′FAM-puromycin incorporation and puromycin signal in amyloid bodies without affecting their biogenesis. In contrast, incubation of cells with a c-myc transcription factor inhibitor had no effect on amyloid-body translational capacity under similar conditions (SI Appendix, Fig. S5C). RNA sequencing and qPCR analysis of purified amyloid bodies confirmed that HSF1-transcribed mRNAs accumulate in these solid-like condensates during stress (Fig. 5 D and E and SI Appendix, Fig. S5 F and G). RNA–fluorescent in situ hybridization (FISH) revealed the presence of Hsp70 mRNA in amyloid bodies, but not in nucleoli, a finding that was dependent on de novo transcription by HSF1 and rIGSRNA. (Fig. 5 F and G). It is interesting that depletion of chromatin-associated rIGSRNA mainly reduces heat shock-induced accumulation of Hsp70 protein in the nucleus rather than cytoplasmic steady-state pools (Fig. 5 H and I). This trend was also observed for other HSF1 targets (SI Appendix, Fig. S5 H and I). However, these experiments do not preclude the possibility that rIGSRNA depletion affects cytoplasmic translation of proteins that are eventually imported into the nucleus (66). Finally, DRB or small interfering RNA (siRNA) against HSF1 prevented the recruitment of the LNPS machinery to amyloid bodies (Fig. 5 J and K and SI Appendix, Figs. S3F and S5 J–M). Pretreatment with cycloheximide, which does not affect amyloid-body biogenesis under our experimental settings, did not prevent such recruitment, indicating that the preexisting translation factors and Rps enter the nucleus to sustain LNPS (SI Appendix, Fig. S5N). These results suggest that newly transcribed HSF1 transcripts are required for LNPS and that these mRNAs recruit the LNPS machinery to drive stress-resistant nuclear translation by amyloid bodies.

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.

Discussion

In this report, we provide evidence that nuclear solid-like condensate amyloid bodies are hubs of stress-resistant protein synthesis. Local nuclear translation relies on the expression of inducible ribosomal intergenic spacer lncRNAs that construct amyloid bodies and on activation of the HSF1 transcriptional program. Our study suggests that amyloid bodies concentrate cytoplasmic translation factors and regulatory Rps to sustain nuclear protein synthesis. Stimuli-induced LNPS joins local translation in neuronal axons (33–36, 67–69) and the rough endoplasmic reticulum (70, 71), highlighting the ability of cells to localize protein synthesis at distinct subcellular domains. These data also indicate that solid-like condensates coordinate complex biochemical reactions similar to their liquid-like counterparts (11, 22, 27, 29).

The central dogma of eukaryotic biology is clear: DNA replication and transcription are nuclear events while protein synthesis occurs in the cytoplasm (72). While this view is deeply entrenched in textbooks, it has undergone frequent challenges over the past few decades by several groups reporting the existence of protein synthesis in the nucleus (47, 73–81). The rather provocative presence of several participants of the translational apparatus in nucleoli has led investigators to propose the existence of nuclear protein synthesis under certain conditions (81). Despite these studies, the “concept of nuclear translation has engaged many but convinced few” as elegantly stated by Reid and Nicchitta (ref. 82, p. 7). There are unanswered questions that have precluded the general acceptance of nuclear protein synthesis (77, 83, 84); many of these questions have been resolved for local translation in axons (34, 85, 86). This study addresses several of these concerns by identifying the following: 1) physiological contexts that enhance nuclear translation (e.g., anaerobic fermentation); 2) key cytoplasmic ribosomal proteins and translation factors that relocalize to amyloid bodies to participate in LNPS; 3) nuclear translating ribosomes with an in vitro assay that excludes the possibility of cytoplasmic contaminants (e.g., cytoplasmic puromycylated peptides); 4) the transcriptional response that drives nuclear protein synthesis (HSF1); and 5) inducible long noncoding RNA (rIGSRNA) that coordinate LNPS. We also note that similar results were obtained with cellular (emetine-treated) and in vitro puromycylation as well as proximity ligation assays across many experimental conditions (e.g., depletion of rIGSRNA, eIF4H, HSF1, etc.). Since the half-life of released puromycylated peptides is very short, the residual ribosome-bound puromycylated peptides may be easier to visualize, especially if concentrated in well-defined foci (e.g., amyloid bodies) and as long as cells are treated with a denaturing fixative (e.g., methanol) for access to the puromycin epitope (45, 87–90). A caveat of using a denaturing fixative is the possibility of generating false proximity between molecules. Nonetheless, silencing of chromatin-associated rIGSRNA that blunts nuclear translation and proximity between LNPS participants/puromycylated peptides with little effect on cytoplasmic protein synthesis further highlights the nuclear origin of LNPS. Likewise, treatment with DRB or depletion of HSF1 prevents the relocalization of LNPS participants to amyloid bodies and nuclear puromycylation reactions. However, our experiments do not preclude that depletion of translation factors (e.g., eIF4H), HSF1, or rIGSRNA has an indirect effect on nuclear translation by altering the synthesis of LNPS participants in the cytoplasm. Interestingly, electron microscopy has revealed that translation-competent nucleoli isolated from liver display the distinct fiber-like morphology (91) that characterizes amyloid bodies, consistent with the observation that these solid-like condensates are present in many intact tissues (19). As prolonged exposure to physiological stimuli is believed to inhibit major nuclear/cytoplasmic trafficking pathways (92–97), it is conceivable that large preassembled ribosomes cannot be efficiently exported to the cytoplasm. Likewise, nuclear import of proteins synthesized in the cytoplasm is impaired during stress. In these settings, it may be advantageous for stressed cells to simply import a few small regulatory Rps/translation factors to complete the assembly of functional ribosomes and produce proteins specific for the nuclear environment while guarding the genome against mis-folded cytoplasmic proteins. As such, our data agree with the local translation field and the necessity to synthesize and concentrate proteins at specific subcellular coordinates under various conditions.

The data shown here provide physiological and mechanistical contexts that will serve as a seed for future work to better understand stress-induced LNPS. We envision that stress-resistant local nuclear translation will play pivotal roles in several physiological and pathological settings such as development, cancer, and ischemic episodes that activate the HSR, among other adaptive pathways. Understanding the exact function of LNPS participants (e.g., eIF4H, eIF4B, etc.) and how mRNAs are targeted to amyloid bodies to drive translation will provide an exciting opportunity to further characterize the nuclear protein synthesis machinery and the role of solid-like condensates in coordinating biochemical reactions.

Materials and Methods

Cell Culture and Reagents.

Human cell lines purchased from the American Type Culture Collection were used in this study; i.e., MCF7, U87MG, and WI-38 were propagated in Dulbecco’s Modified Eagle Media (DMEM) (HyClone) with 10% fetal bovine serum (Omega Scientific) and 1% penicillin–streptomycin (HyClone). Cells were maintained at 37 °C in a 5% CO₂ humidified incubator (normoxia neutral, NN). Cells were subjected to hypoxia (hypoxia neutral, HN) (1% O₂, 24 h, unless otherwise stated) at 37 °C in a 5% CO₂, N₂-balanced, humidified H35 HypOxystation (HypOxygen). Cells were subjected to hypoxia acidosis by culturing them in acidotic permissive media in the hypoxic chamber. Cells were introduced to DMEM without bicarbonate at pH 7.4. They were allowed to naturally acidify to a pH of ∼6.0 (∼30 min). Cells were subjected to heat shock by translocation to a 5% CO₂ humidified incubator maintained at 43 °C and incubated there for a minimum of 2 h and maximum of 6 h. Thermal stress of 4 to 6 h was used for puromycin experiments in intact cells and localization of factors by immunofluorescence while 2 h heat shock was sufficient for all other experiments. For staining experiments, cells were grown on poly-L-lysine–coated coverslips (Neuvitro). Puromycin (ThermoFisher Scientific) was used at a final concentration of 2 μM for the last 10 min of stimulus. 6′FAM-puromycin (Jena Bioscience, Lot BM015-073) was used at a final concentration of 20 μM for 10 min at 4 °C, diluted in Polysome Buffer (50 mM Tris⋅HCl, 5 mM MgCl₂, 25 mM KCl, ethylenediaminetetraacetic acid (EDTA)-free protease inhibitors, 10 U/mL RNase out). Anisomycin (Sigma) and ampicillin (Sigma) were used at indicated competing molarities. Emetine (Sigma) was used at a final concentration of 200 μM and added 5 min prior to incubation with puromycin or 5 min prior to solubilization for in vitro FAM-puromycylation. Cyclohexamide (Sigma) was used at a final concentration of 0.2 mg/mL and added 5 min prior to incubation with puromycin. Harringtonine (MedChemExpress) was used at a final concentration of 2 μg/mL while aurintricarboxylic acid (Sigma) was used at a final concentration of 100 uM, both of which were added 20 min prior to incubation with puromycin. DRB (Sigma) was administered 15 min prior to stimulus at a final concentration of 100 μM. RNase (ThermoFisher Scientific) treatments of 100 μg/mL were used. KRIBB11 (EMD Millipore) was used at a final concentration of 50 μM and added onto cells 30 min prior to heat shock. CCT251236 (Axon Medchem) was used at a final concentration of 200 nM, HSF1A (Axon Medchem) was used at a final concentration of 100 μM, and 10058-F4 (Axon Medchem) was used at a final concentration of 100 μM, all three of which were added to cells 30 min prior to heat shock.

Immunocytochemistry.

Cells were washed once with cold phosphate-buffered saline (PBS), then fixed on coverslips for 10 min in methanol, washed with PBS, and then permeabilized for 10 min using 0.5% Triton-X (in PBS). After wash, cells were blocked with 5% horse serum (in phosphate buffered saline with tween [PBST]) for 30 min. Cells were incubated for 1 h at 37 °C or at 4 °C overnight in primary antibody (1:100) and washed and incubated with corresponding secondary antibody (1:500) for 1 h at 37 °C. Cells were washed three times for 10 min, and nuclei were stained using Hoescht 33258 (1:1,000, ThermoFisher Scientific) during the second wash. Cells were mounted on slides using Fluoromount and visualized and analyzed by fluorescent microscopy with the BZ-X800 High-Resolution Imaging and Analysis System (Keyence). Excitation wavelengths were 334 nm/438 to 533 nm for Amylo-glo, 488 nm/509 to 529 nm for AlexaFluor 488 and anti–DIG-488, and 555 nm/569 to 589 nm for AlexaFluor 555 and Congo red. Images were uniformly adjusted to increase brightness/contrast with Photoshop CC (Adobe). To determine nuclear/cytoplasmic ratio, the corrected total cell fluorescence was determined for both the nucleus and cytoplasm using ImageJ (CTCF = Integrated Density – [area of selected cell × mean fluorescence of background readings]) and compared = CTCF^nucleus/CTCF^cytoplasm. The following primary antibodies were used (all 1:100): puromycin (4G11) (Millipore, MABE342), calreticulin (Invitrogen, PA3-900), Rpl24 (Proteintech, 17082–1-AP), Rps26 (Proteintech, 14909–1-AP), Rpl7 (Proteintech, 14583–1-AP), Rpl27 (Proteintech, 14980–1-AP), RplP0 (Proteintech, 11290–2-AP), Rps9 (Proteintech, 18215–1-AP), Rps3 (Proteintech, 66046–1-Ig), Rps6 (Cell Signaling, #2217), eIF4H (Cell Signaling, #3469), eIF4A1 (Abcam, ab31217), methionyl tRNA synthetase (MARS) (Abcam, ab50793), arginyl tRNA synthetase (Novus, NBP1-46148), lysyl tRNA synthetase (Abcam, ab31532), eEF1E1 (Novus, H00009521-B01P), eIF1 (Proteintech, 14654–1-AP), eIF2A (Proteintech, 11233–1-AP), eIF2α (Cell Signaling, #5324), eIF2D (Proteintech, 12840–1-AP), eIF2S1 (Abcam, ab32157), eIF3D (Abcam, ab155419), eIF3E (Abcam, ab36766), eIF3F (Abcam, ab64177), eIF3K (Santa Cruz, sc-81262), eIF4A2 (Proteintech, 11280–1-AP), eIF4A3 (Proteintech, 17504–1-AP), eIF4B (Novus, NB100-93308), eIF4E1 (Abcam, ab1126), eIF4E2 (Genetex, GTX103977), eIF4E3 (Proteintech, 17282–1-AP), eIF4G1 (Novus, NB100-268), eIF4G2 (Cell Signaling, #5169), eIF4G3 (Genetex, GTX118109), eIF5 (Santa Cruz, sc-135894), eIF5A1 (Abcam, ab32443), eIF5A2 (ThermoFisher Scientific, PA5-30770), eIF5B (Santa Cruz, sc-393564), eIF6 (Santa Cruz, sc-390432), B23 (Santa Cruz, sc-32256), Cdc73 (ThermoFisher Scientific, PA5-26189), Hsp70 (Santa Cruz, sc-66048), and Hsp70 n-term (Aviva, ARP74952_P050).

Immunocytochemistry: Puromycin Assay in Intact Cells.

Cells grown on coverslips were treated with emetine at a final concentration of 200 μM 5 min prior to the dropwise addition of puromycin per each condition. After 10 min of puromycin treatment, cells were washed once with cold PBS. Fixation was achieved with a 10-min incubation of cold methanol which acts by dehydrogenation and protein precipitation and can reveal the puromycylated peptides that would otherwise be sterically hidden in translating ribosomes. Cells were washed with PBS and then permeabilized for 10 min using 0.5% Triton-X (in PBS). After wash, cells were blocked with 5% horse serum (in PBST) for 30 min, and antibody staining was performed as described above.

In Vitro 6′FAM-Puromycin Assay.

Cells grown on coverslips were removed from each condition and washed with cold polysome buffer (50 mM Tris⋅HCl, 5 mM MgCl₂, 25 mM KCl, EDTA-free protease inhibitors, 10 U/mL RNase out). From this point onward, the assay was performed at 4 °C, a temperature at which few nascent chains are released (51, 53, 54). Cells were incubated for 15 min with permeabilization buffer (50 mM Tris⋅HCl, 5 mM MgCl₂, 25 mM KCl, EDTA-free protease inhibitors, 10 U/mL RNase out, 0.1% Nonidet P-40) and then washed gently with polysome buffer twice. Cells were incubated for 10 min with 20 uM 6′FAM-puromycin (Jena Bioscience, Lot BM015-073) diluted in polysome buffer in a light-protected manner. Cells were washed 3× with polysome buffer and fixed with 1% formaldehyde for 10 min. Cells were washed with polysome buffer again, stained with Hoescht 33258, and mounted for visualization as described above.

Propidium Iodide and Fluorescein Diacetate Staining.

Live cells were incubated with final concentration of 1 μg/mL each of propidium iodide, fluorescein diacetate, and Hoescht 33342 (ThermoFisher Scientific) for 30 min at 37 °C and washed twice with media before imaging by fluorescence microscopy. Cell counting was done manually or via ImageJ for viability measurements.

Nuclear/Cytoplasmic Extraction.

Cells were washed with cold PBS and harvested in PBS and then kept on ice for the remaining procedure. Microcentrifuge tubes containing harvested cells were centrifuged briefly for 5 to 10 s at 10,000 × g to pellet cells. Supernatant was removed, and cells were resuspended in cold PBS containing 0.1% Nonidet P-40 and then triturated five times on ice with a P1000 pipette tip. A fraction of this solution was set aside as whole-cell lysate. Remaining cell suspension was centrifuged briefly for 5 to 10 s at 10,000 × g. A portion of the supernatant was set aside as cytoplasmic fraction. The remaining supernatant was discarded, removing drops with an extended-length P100 pipette tip. Pellet was resuspended in PBS containing 0.1% Nonidet P-40 and then centrifuged briefly for 5 to 10 s at 10,000 × g. Supernatant was discarded, removing drops with an extended-length P100 pipette tip. Nuclear pellet was washed three times with cold PBS and then resuspended in Ripa buffer (ThermoFisher Scientific). Nuclear fractions were sheared with 25-g needles (BD) and centrifuged at maximum speed for 10 min to remove DNA.

Immunoblot.

Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed on Bolt 4 to 12% Bis-Tris Plus premade gels (ThermoFisher Scientific) using the Mini Gel Tank system (ThermoFisher Scientific) and transferred to 0.2- μm Immuno-Blot PVDF membranes (Bio-Rad) using the Bolt Mini Blot Module (ThermoFisher Scientific), all according to the manufacturer’s protocols. Chemiluminescent signals were detected using SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific) on an Amersham Imager 600 (G9E Healthcare Life Sciences). Primary antibodies used (all 1:1,000) were the following: puromycin (Kerafast, EQ0001), calreticulin (Invitrogen, PA3-900), β-actin (Santa Cruz, sc-47778), Rps3 (Proteintech, 66046–1-Ig), Rpl24 (Proteintech, 17082–1-AP), Rps26 (Proteintech, 14909–1-AP), Cdc73 (ThermoFisher Scientific, PA5-26189), fibrillarin (Santa Cruz, sc-25397), GAPDH (Santa Cruz, sc-47724), eIF4H (Cell Signaling, #3469), eIF4A1 (Abcam, ab31217), Hsp40 (DNAJB1) (Proteintech, 13174–1-AP), Hsp70 n-term (Aviva, ARP74952_P050), eIF2A (Proteintech, 11233–1-AP), eIF4A2 (Proteintech, 11280–1-AP), eIF4B (Novus, NB100-93308), eIF5A1 (Abcam, ab32443), eIF5A2 (ThermoFisher Scientific, PA5-30770), HSF1 (Santa Cruz, sc-17757), METTL3 (Proteintech, 15073–1-AP), and YTHDF2 (Proteintech, 24744–1-AP). Amyloid-body purification for immunoblotting was performed as previously reported by Audas et al. (19).

Amylo-Glo Staining.

Cells were washed with cold PBS and fixed with 4% formaldehyde for 10 min. Cells were washed with PBS and permeabilized with 0.5% Triton-X (in PBS) for 5 min. After 2 min of still incubation with nuclease-free water, cells were incubated with Amylo-glo (1:100, Biosensis) in 0.9% saline for 10 min. Following 15 s of rinsing with nuclease-free water, coverslips were mounted with 5% glycerol and sealed with varnish.

Congo Red Staining.

Cells were fixed with 4% formaldehyde for 10 min and then permeabilized with 0.5% Triton for 10 min. Cells were washed with PBS and then stained with Hoescht 33258 (1:1,000) for 10 min. Cells were washed with ddH20 and then incubated with Congo Red solution (3.5 mM Congo Red, 0.5 M NaCl, 80% EtOH) for 15 min, washed three times with ddH20, and mounted with 5% glycerol for visualization.

Proximity Ligation Assay.

Cells were grown at basal conditions or heat shocked for 4 h. For siRNA experiments, cells were heat shocked for 2 h. Emetine was added 15 min prior to the end of treatment. At 10 min prior to the end of treatment, puromycin was added. At the end of treatment, cells were washed in PBS, fixed in cold methanol for 10 min, and permeabilized with 0.5% Triton X in PBS. Cells were probed with antibodies for eIF4h, eIFA1, Rps3, and Rps26, and puromycin. For negative control experiments, samples were incubated without puromycin antibody. For in vitro PLA, cells were prepared as outlined in Fig. 1B. Primary antibody incubation was 16 h at 4 °C, and PLA was performed according to Duolink In Situ PLA protocol. The following reagents used were: DUO92005-30RXN Duolink In Situ PLA Probe Anti-Rabbit MINUS, DUO92001-30RXN Duolink In Situ PLA Probe Anti-Mouse PLUS, DUO92008-30RXN Duolink In Situ Detection Reagents Red, DUO82049-4L Duolink In Situ Wash Buffers, fluorescence, and DUO82040-5ML Duolink In Situ Mounting Medium with DAPI.

RNA Interference and Transfections.

Target-specific pools of four independent siRNA species (siGENOME SMARTpool, Dharmacon) were purchased for most targets. siRNAs against rIGSRNA were constructed by ThermoFisher Scientific. siRNAs (100 pmol) were reverse-transfected using RNAiMAX (Thermo Fisher Scientific) and treated/harvested 48 h post transfection.

MS-SILAC.

For each condition shown, MCF‐7 cells were grown in stable isotope labeling with amino acids in cell culture (SILAC)‐labeling media, amyloid‐body–containing fractions were purified, and samples were analyzed by mass spectrometry (MS) following previously described protocols. MS datasets have been published in Audas et al. (19) and Marijan et al. (24). Raw ratios were used for the proteomic output analysis of ribosomal proteins in nucleoli versus amyloid bodies from heat-shocked or acidotic cells, one-fold enrichment signifying no change between basal and stress treatment.

RNA Sequencing.

Cells were either grown at basal conditions or heat shocked for 1 h. Plates were then washed with PBS, and cells were harvested in cold solution 1 (0.5 M sucrose, 3 mM MgCl₂) and sonicated for 6 × 10 s using a microtip probe. Samples were then underlaid with cold solution 2 (1 M sucrose, 3 mM MgCl₂) and centrifuged at 2,800 × g for 10 min at 4 °C. Pellets were resuspended in PBS, and a fraction of each sample was used to determine proper isolation of nucleoli and amyloid bodies by microscopic evaluation. Remaining resuspended samples were then used for RNA isolation with TRIzol reagent (Invitrogen) and submitted for total RNA sequencing.

qRT-PCR.

First-strand complementary DNA (cDNA) synthesis was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), according to the manufacturer’s protocols. qRT-PCR was performed using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Relative changes in expression were calculated using the comparative Ct (ΔΔCt) method. Primer sequences are available upon request.

Fluorescent In Situ Hybridization.

FISH was performed with 5′ and 3′ digoxigenin (DIG)-labeled oligonucleotides. Following 30 min fixation, 0.1 M Tris⋅HCl, pH 7.0 was added to cells for 10 min before ± Proteinase K treatment (NEB, 800 U/mL stock, 100,000× dilution) at 37 °C for 30 min. Cells were equilibrated in 2× saline–sodium citrate buffer (SSC) before overnight hybridization at 37 °C. Probes (10 pmol) were denatured at 85 °C for 10 min. Hybridization buffer was 15% formamide, 10% dextran sulfate, 2 mM vanadyl ribonucleoside, and 2× SSC. Probes were detected with an anti–DIG-flourescin antibody (Sigma, 11207750910) at 20 μg/L in 4× SSC. Slides were mounted in 90% glycerol. The following RNA-FISH probes were used: Hsp70 (TGC​TGA​AAC​ACG​CCC​ACG​CAC​GAG​TAG​GTG​GTG​CCC​AGG​TCG​ATG​CCC​AC) and HspA1A (GAC​AAC​GGG​AGT​CAC​TCT CGA​AAA​AGG​TAG​TGG​ACT​GTC​GCA​GCA​GCT​CC).

Statistical Analysis.

All experiments were performed at least two independent times, unless otherwise stated. Quantitation of microscopy-based data was performed using ImageJ on at least three representative images. Graphs represent mean values, and error bars depicted represent SD of the mean (SEM) between repeats. Appropriate statistical analyses were performed (e.g., Student’s t test), while significance was defined as P < 0.05.

Data Availability.

RNA-sequencing data have been deposited in the Gene Expression Omnibus database (accession no. GSE164846) (98).