Local translation in nuclear condensate amyloid bodies Phaedra R. Theodoridisa,b,1, Michael Bokrosa,b,1, Dane Marijanc, Nathan C. Balukoffa,b, Dazhi Wanga,b, Chloe C. Kirka,b, Taylor D. Budinea,b, Harris D. Goldsmitha,b, Miling Wanga,b, Timothy E. Audasc, and Stephen Leea,b,2 aDepartment of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL 33136; bSylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33136; and cDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6 Edited by Peter Cresswell, Yale University, New Haven, CT, and approved January 11, 2021 (received for review July 9, 2020) Biomolecular condensates concentrate molecules to facilitate basic similar to Balbiani bodies in Xenopus oocytes (31). In contrast to biochemical processes, including transcription and DNA replica- liquid-like condensates that concentrate molecules to facilitate tion. While liquid-like condensates have been ascribed various biochemical reactions, amyloid bodies and Balbiani bodies are functions, solid-like condensates are generally thought of as amor- believed to store proteins and induce cellular dormancy (19, 27, phous sites of protein storage. Here, we show that solid-like am- 31). Since amyloid bodies also comprise an array of mobile yloid bodies coordinate local nuclear protein synthesis (LNPS) proteins (e.g., ribosomal proteins) (23), it remains unknown if during stress. On stimulus, translationally active ribosomes accu- these nuclear condensates have other cellular functions in ad- mulate along fiber-like assemblies that characterize amyloid bod- dition to protein retention and metabolic depression. ies. Mass spectrometry analysis identified regulatory ribosomal Here, we report that nuclear amyloid bodies are sites of stress- proteins and translation factors that relocalize from the cytoplasm resistant local nuclear protein synthesis (LNPS). Mass spec- to amyloid bodies to sustain LNPS. These amyloidogenic compart- trometry with stable isotope labeling by amino acids in cell culture ments are enriched in newly transcribed messenger RNA by Heat (MS-SILAC) cytoplasmic ribosomal proteins and translation fac- Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal inter- tors that relocalize to amyloid bodies in cells responding to an- genic spacer noncoding RNA (rIGSRNA) that constructs amyloid aerobic fermentation or heat shock. In vitro puromycylation and bodies prevents recruitment of the nuclear protein synthesis ma- proximity ligase assays uncovered ribosomal acceptor sites orga- chinery, abolishes LNPS, and impairs the nuclear HSF1 response. nized along fiber-like assemblies that characterize these solid-like We propose that amyloid bodies support local nuclear translation condensates. Amyloid bodies anchor HSF1 target mRNAs that during stress and that solid-like condensates can facilitate complex CELL BIOLOGY biochemical reactions as their liquid counterparts can. recruit the LNPS machinery to sustain nuclear protein synthesis. We discuss how amyloid-body protein synthesis can be compared long noncoding RNA | hypoxia | acidosis | HSR | Hsp70 to local translation machineries that synthesize proteins at specific cellular coordinates (32–40) during periods of inhibition of nu- clear/cytoplasmic trafficking. These results identify amyloid bodies ells are remarkable in their ability to sustain viability under as nuclear, solid-like condensates that sustain stress-induced local Cadverse environmental conditions (1). Hypoxia-induced ex- tracellular acidosis is a common stimulus observed in an array of translation. physiological and pathological settings including development, Results cancer, and ischemic episodes (2, 3). Cells acclimatize to the acidotic state by suppressing global protein synthesis among Physiological Stressors Induce LNPS in Amyloid Bodies. Cells were other pathways (4–6) while activating the heat shock response incubated in low-oxygen tension to induce the natural acidifi- (HSR) involved in protein homeostasis (7–9). The master regulator cation of their extracellular milieu as a consequence of lactic of the HSR is heat shock factor 1 (HSF1) that transcribes genes encoding a wide array of chaperones, including the canonical heat Significance shock protein 70 (Hsp70) (10). Activation of the HSR through HSF1 is believed to sustain cellular viability during periods of intense stress, The central dogma of eukaryotic biology is clear: DNA replica- such as extracellular acidosis and heat shock (9). tion and RNA transcription occur in the nucleus while protein In addition to activating the HSR, extracellular acidosis and synthesis takes place in the cytoplasm. While this fundamental heat shock induce the formation of several membraneless bodies tenet of biology is entrenched in textbooks, it has undergone (e.g., stress granules) that have been implicated in a wide range recurring challenges over the decades, most notably the exis- of cellular functions, such as storage of cytoplasmic messenger tence of nuclear translation. We report that solid-like conden- RNAs (mRNAs) and nuclear splicing factors. These organelles sate amyloid bodies are hubs of stress-resistant local nuclear have been described as liquid-like biomolecular condensates and translation in cells engaging in hypoxic fermentation or are formed by a phenomenon called “phase separation” (11–14). responding to high temperature. Conceptually, this paper Liquid-like biomolecular condensates are typically dynamic as provides a physiological context for nuclear protein synthesis their constituent proteins are highly mobile and can exchange while highlighting a role for solid-like condensates in coordi- between the phase-separated bodies and the extracellular milieu nating complex biochemical reactions. (15–18). Fermentation-induced acidosis and heat shock also stimulate the formation of amyloid bodies: nuclear organelles Author contributions: P.R.T., M.B., T.E.A., and S.L. designed research; P.R.T., M.B., D.M., that can be readily distinguished from other compartments by N.C.B., D.W., C.C.K., T.D.B., H.D.G., and M.W. performed research; P.R.T., M.B., N.C.B., their large electron-dense fibers that stain with various amyloi- T.E.A., and S.L. analyzed data; and P.R.T., T.E.A., and S.L. wrote the paper. dophylic dyes (19). Amyloid bodies are constructed by a class of The authors declare no competing interest. inducible long noncoding RNA (lncRNA) derived from stimuli- This article is a PNAS Direct Submission. specific loci of the ribosomal intergenic spacer (rIGSRNA) (20–22). Published under the PNAS license. Mass spectrometry and photobleaching analyses revealed that 1P.R.T. and M.B. contributed equally to this work. amyloid bodies are enriched in a heterogeneous population of 2To whom correspondence may be addressed. Email: [email protected]. full-length proteins, many of which are immobile (23–26). As This article contains supporting information online at https://www.pnas.org/lookup/suppl/ such, amyloid bodies have been described as nondynamic, or doi:10.1073/pnas.2014457118/-/DCSupplemental. solid-like, condensates with amyloidogenic properties (27–30) Published February 10, 2021. PNAS 2021 Vol. 118 No. 7 e2014457118 https://doi.org/10.1073/pnas.2014457118 | 1of9 Downloaded by guest on September 24, 2021 fermentation, recapitulating various in vivo conditions including ribosomes (51, 53, 54). Consistent with previous reports (47), a ischemia (41) and microenvironments of aggressive tumors (42). slightly over background nuclear 6′FAM-puromycin signal was Under such anaerobic acidosis conditions (37 °C, 1% O2, pH observed in permeabilized cells that were maintained at basal 6.0), cells remain viable while displaying a marked decrease in conditions (Fig. 1C: first panel and fourth panel for low magni- global translation compared to their basal (37 °C, 21% O2, pH fication). In sharp contrast, intense nuclear 6′FAM-puromycin 7.4) or anaerobic neutral (37°, 1% O2, pH 7.4) counterparts (SI signal was detected in acidotic or thermal-stressed cells (Fig. 1C: Appendix, Fig. S1 A and B). To corroborate these results, we second and third panels and fifth panel for low magnification; performed immunofluorescence analysis on cells incubated with Fig. 1D). Anisomycin competes with puromycin for ribosomal puromycin (SI Appendix, Fig. S1C). Cells were treated with A-site binding although these molecules are structurally different emetine to prevent the synthesis of new proteins and fixed with and thus unlikely to bind to the same nonspecific sites (47, 55, the denaturant methanol to enable the detection of free and 56). Addition of anisomycin abolished in vitro 6′FAM- residual translating, ribosome-associated, puromycylated pep- puromycin incorporation (Fig. 1 C and D and SI Appendix, Fig. tides (43, 44). Under these experimental conditions, we observed S1 F and G), demonstrating that the puromycylation reactions abundant puromycin signal in the cytoplasm of cells maintained occur at bona fide ribosomal A-sites in the nucleus. Likewise, at basal (37 °C, 21% O2, pH 7.4) or hypoxic neutral (37°, 1% O2, addition of anisomycin abolished puromycin signal in intact cells pH 7.4) conditions (Fig. 1A). Surprisingly, puromycin signal was (Fig. 1A). The presence of nuclear ribosomes actively synthe- detected in nuclear foci, in addition to the cytoplasm, in cells sizing proteins was further demonstrated by the detection of engaging in anaerobic