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RNA Impacts Formation of Biomolecular Condensates in the Nucleus

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RNA Impacts Formation of Biomolecular Condensates in the Nucleus Biomedical Research (Tokyo) 42 (4) 153–160, 2021 RNA impacts formation of biomolecular condensates in the nucleus Saho MATSUI and Ryu-Suke NOZAWA Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Ariake 3-8-31 Koto-ku, 135-8550 Tokyo, Japan (Received 10 May 2021; and accepted 11 June 2021) ABSTRACT Biomolecular condensates are membrane-less compartments that are formed through an assembly of proteins and nucleic acids in the cell. Dysregulation of biological condensates has been impli- cated in diseases such as neurodegeneration and cancer. Ribonucleic acid (RNA) is known to af- fect the assembly of proteins in vitro, if and how RNA is involved in regulating biomolecular condensates in cells is not well investigated. Here we examined two nuclear proteins, FUS and HP1α, in which RNA was found to have an opposite contribution for the assembly of these pro- teins. Reduction of nuclear RNA, by inhibiting the transcription, triggered assembly of FUS that had been distributed in the nucleoplasm, whereas it dispersed spontaneously formed HP1α assem- bly. Notably, the cell cycle-dependent phosphorylation-mimicking substitutions in HP1α promoted its assembly formation. These transcription inhibitor experiments are versatile to examine diverse roles of nuclear RNA in regulating biomolecular condensates, in both physiological and pathologi- cal conditions. uid phase composed of multiple soluble ingredients INTRODUCTION separating into different phases through their selec- Cells organize compartments in their interior to dif- tive and exclusive interactions (as a salad dressing ferentiate and facilitate biological processes. The nu- separates into oil- and water-phases). Through an cleus and mitochondria contain a lipid bilayer to analogous mechanism, selective assembly of bio- physically separate inside and outside and maintain molecules, such as proteins and nucleic acids, drives their internal biochemical environment. Another type the formation of distinct compartments inside the of cellular compartment including nucleoli, promy- cell. These what it is termed “biomolecular conden- elocytic leukaemia (PML) bodies involved in ge- sates” are based on labile multivalent interactions nome maintenance, Cajal bodies and P granules in between proteins and/or nucleic acids, e.g., the elec- RNA metabolism, and stress granules are formed trostatic interaction between the charged amino ac- without a physical barrier. The principle that under- ids of proteins and the negatively charged phosphate lie this compartmentalization is explained by the backbone of nucleic acids. Thus, biomolecular con- phenomenon called liquid-liquid phase separation, densates result from the selective attractions be- or LLPS (Banani et al. 2017; Shin and Brangwynne tween their components to enrich the components in 2017). a distinct space. LLPS is a physical property in which a single liq- Dysregulation of biomolecular condensates is asso- ciated with various pathological conditions including Address correspondence to: Ryu-Suke Nozawa, Divi- cancer and neurodegenerative diseases like amyo- sion of Experimental Pathology, The Cancer Institute of trophic lateral sclerosis (ALS) and frontotemporal JFCR, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan lobar degeneration (FTLD) (Aguzzi and Altmeyer Tel: +03-3570-0445, Fax: +03-3570-0354 2016; Alberti and Dormann 2019; Nozawa et al. E-mail: [email protected] 2020). Solid aggregates of RNA binding proteins in- 154 S. Matsui and R. Nozawa cluding FUS (fused in sarcoma) have been observed mL) and actinomycin D (50 ng/mL) for 5 h. in the neurons of ALS patients, which is thought to causally relate to their degeneration (Kwiatkowski et Plasmids and plasmid transfection. cDNA for human al. 2009; Vance et al. 2009). FUS primarily localiz- FUS is a gift from Takuya Yoshizawa (Ritsumeikan es in the nucleus and forms biomolecular conden- University). cDNA for human HP1α is generated sates to promote transcription and DNA damage from hTERT-RPE1 cell mRNA by reverse transcrip- repair (Altmeyer et al. 2015; Wei et al. 2020). How- tion PCR. Point mutations of HP1α were construct- ever, the cytoplasmic mis-localized FUS, caused by ed by a standard PCR cloning strategy. All cDNAs its mutations or other conditions, triggers an aber- were inserted into a modified pcDNA5/FRT expres- rant transformation into the FUS aggregates. sion vector (ThermoFisher) with an N-terminal What is the difference in environment between the mEGFP tag. mEGFP is a monomeric enhanced GFP nucleus and the cytoplasm? One apparent difference (EGFP) variant having Ala substitution at Lys 206. is the concentration of RNA. After transcription, HP1α E-E has seven Glu substitutions at Ser 11, Ser only a small fraction is exported to the cytoplasm 12, Ser 13, Ser 14, Ser 92, Ser 95 and Ser 97. HP1α for translation and a large amount of RNA remains E-A has four Glu substitutions at Ser 11, Ser 12, Ser in the nucleus (Nozawa and Gilbert 2019). It is esti- 13, and Ser 14, and three Ala substitutions Ser 92, mated that the concentration of RNA in the nucleus Ser 95 and Ser 97. HP1α A-A has seven Ala substi- is approximately 36 times higher than that in the cy- tutions at Ser 11, Ser 12, Ser 13, Ser 14, Ser 92, Ser toplasm (Maharana et al. 2018). Besides being fun- 95 and Ser 97. All plasmids were verified by se- damental elements for transcription and translation, quencing. Plasmid transfection was carried out by another role of nuclear RNA is to regulate global incubating cells in a 6 well plate with 2 μg plasmids chromatin structures by forming a mesh-like struc- with 4 μL FuGENE HD (Promega) for 24 h. ture with scaffold attachment factor A (SAF-A) (also called heterogeneous nuclear ribonucleoprotein Imaging analysis. Cells were fixed with 2% paraform- (hnRNP U) (Nozawa et al. 2017; Nozawa and Gilbert aldehyde in PBS for 10 min at room temperature (rt), 2019). Nuclear RNA is also involved in protein as- followed by incubation in PBS containing 0.2% Tri- sembly through electrostatic interactions by virtue of ton X-100 for 10 min at rt. DNA was then stained its negatively charged phosphate backbone (Zhang with 0.1 μg/mL DAPI. After staining, cells were et al. 2015; Maharana et al. 2018). These observa- mounted with ProLong Gold anti-fade mounting re- tions exemplify the role of RNA in interfering with agent (Invitrogen). Images were acquired on an the formation of condensates, while less is known LSM 880 confocal laser scanning microscope (Zeiss) whether RNA serves as a seed to attract RNA bind- using 63× objective lens (1.4. NA Plan Apochromat ing proteins to nucleate assemblies in the nucleus. Oil DIC M27) with immersion oil (Zeiss Immersol Here we examined if decreased levels of nuclear 518F; refractive index 1.518). Scale of x, y was set RNA affect protein assembly activities of FUS and at 0.053, 0.053 μm/pixel, respectively, and scan heterochromatin protein 1α (HP1α) in the nucleus. speed was typically 1 μs/pixel. Seven images per Our observations indicate the contribution of nucle- cell were acquired at 0.5-μm intervals and were ar RNA in preventing the self-assembly of FUS and Z-stacked with maximum intensity projection (Image in promoting the formation of the HP1α-assembly. J, version 2.1.0/1.53c). The area of the HP1α assem- Interestingly, the cell cycle-dependent phosphoryla- bly was quantified for sizes larger than 0.05 μm2 tion of HP1α revealed a significant effect on their with Image J. For the analysis of HP1α disassembly nuclear assembly. We propose that transcription in- with transcription inhibition, 3 images per cell were hibitor experiment is a versatile method to examine captured every 5 min for 5 h with a 60× objective the involvement of nuclear RNA in the formation of lens, at 0.5-μm intervals, 1% of 488 laser excitation, biomolecular condensate. 50 msec exposure time on CellVoyager CV1000 (Yokogawa) and Z-stacked with maximum intensity projection (Image J, version 2.1.0/1.53c). MATERIAL AND METHODS Cell culture. HEK293T cells were cultured in Dul- RESULTS becco’s modified Eagle’s medium (DMEM) supple- mented with 10% FCS and 10 mM HEPES-NaOH FUS forms assembly in response to transcription in- (pH 7.5) at 37°C in a 5% CO2 environment. Tran- hibition scription was blocked by adding α-amanitin (50 mg/ FUS is an RNA binding protein containing RGG RNA regulates protein assembly 155 Fig. 1 FUS forms assembly in response to transcription inhibition. (A) Fluorescence microscopy of HEK293T cells ex- pressing mEGFP-FUS or mEGFP, treated with or without α-amanitin and actinomycin D. Scale bar, 10 μm. (B) Quantifica- tion of cells showing the indicated distribution of mEGFP-FUS in the nucleus, with or without α-amanitin and actinomycin D (n > 70 cells for at least two biological replicates). (arginine glycine glycine) RNA-binding motifs and of RNA from FUS and thereby promoted the FUS- is known to have a self-assembly property through FUS self-association. Supporting this interpretation, the LLPS mechanism (Wang et al. 2018). When we the disruption of nuclear RNA by introduction of expressed mEGFP-fused human FUS in HEK293T RNase A, by microinjection, into the nucleus has cells, mEGFP-FUS revealed diffusible distribution been shown to trigger the FUS assembly (Maharana throughout the entire nucleus, but excluded from et al. 2018). These observations suggest that nuclear DAPI low-density nucleoli (Fig. 1A). To examine RNA contributes to preventing spontaneous FUS whether nuclear RNA affects the FUS behavior, we self-assembly. treated these cells with α-amanitin, an RNA poly- merase II (pol II) inhibitor, expecting to reduce the HP1α spontaneously forms nuclear assembly that is level of cellular RNA. The α-amanitin treatment re- enhanced by phosphorylation-mimicking substitu- sulted in an accumulation of mEGFP-FUS in nucle- tions oli, which coincided with its apparent decrease from To further investigate the role of nuclear RNA in the nucleoplasm (Fig.
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