Article

https://doi.org/10.1038/s41467-022-35602-w

PQBP5/NOL10 maintains and anchors thenucleolus under physiological and osmotic
stress conditions
Received: 27 January 2022

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Accepted: 13 December 2022

Xiaocen Jin1,4, Hikari Tanaka 1,4, Meihua Jin1, Kyota Fujita1, Hidenori Homma
Maiko Inotsume1, Huang Yong1, Kenichi Umeda2, Noriyuki Kodera 2,
Toshio Ando 2 & Hitoshi Okazawa 1,3

1,

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10(NOL10), binds to polyglutamine tract sequences and is expressed in the
nucleolus. Using dynamic imaging of high-speed atomic force microscopy, weshow that PQBP5/NOL10 is an intrinsically disordered protein. Superresolution microscopy and correlative light and electron microscopy method
show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus,
constituting the granule meshwork in the granular component area, which isdistinct from other nucleolar substructures, such as the ﬁbrillar center anddense ﬁbrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10
remains in the nucleolus and functions as an anchor for reassembly of othernucleolar proteins. Droplet and thermal shift assays show that the biophysicalfeatures of PQBP5/NOL10 remain stable under stress conditions, explaining
the spatial role of this protein. PQBP5/NOL10 can be functionally depleted bysequestration with polyglutamine disease proteins in vitro and in vivo, leading
to the pathological deformity or disappearance of the nucleolus. Taken
together, these ﬁndings indicate that PQBP5/NOL10 is an essential protein
needed to maintain the structure of the nucleolus.

The nucleolus is a critical structure for the transcription of ribosomal
DNA (rDNA) to ribosomal RNA (rRNA), speciﬁcally 45S pre-rRNA, by
RNA polymerase I. The nucleolus forms around the tandem repeats ofrDNA genes called nucleolar organizing regions (NORs). The 45S prerRNA is processed to 18S, 5.8S, and 28S RNA molecules and assembled
with 5S rRNA transcribed by RNA polymerase III in the nucleoplasm.
Subsequently, these RNA molecules are combined with ribosomal
proteins (RPs) in the nucleus and cytoplasm to form 60S and 40S
ribosomes. Because the protein translation activity of ribosomes isessential for the maintenance, differentiation, and stress responses ofcells, the nucleolus is a master subcellular structure that regulates cellfunctions and phenotypes.
Because many components of the nucleolus, such as ﬁbrillarin andnucleolin, have been recognized as intrinsically disordered proteins(IDPs) containing low complexity sequences, the nucleolus is now
considered a liquid-liquid phase separation (LLPS) droplet1–12. Substructures of the nucleolus include a ﬁbrillar center (FC), a dense ﬁbrillar
component (DFC), and a granular component (GC). The FC and DFC arefunctionally linked to rRNA transcription by RNA polymerase I located at
the interface between the FC and the DFC, whereas the functional role of1

Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 2Nano
Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. 3Center for Brain Integration Research, Tokyo Medical and
Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 4These authors contributed equally: Xiaocen Jin, Hikari Tanaka.
e-mail: okazawa-tky@umin.ac.jp

Nature Communications | (2023)14:9

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the GC remains unclear. The GC region contains the negatively charged
molecules nucleophosmin (NPM1) and nucleolin10,13. NPM1 homotypicassembly forms the boundary between the nucleolus and nucleoplasm
and may contribute to ribosome maturation5.
Various cell stresses, including DNA damage, temperaturechange, hypoxia, viral infection, serum starvation, and transcriptional
suppression, have been found to affect the morphology and functionsof nucleoli14. Osmotic stress has been associated with irregular deformation of the nucleolus, although the detailed molecular mechanisms
accounting for these morphological changes remain unclear15. Osmotic stress has also been reported to affect another nuclear substructure, Cajal bodies, although that study did not investigate
nucleolar changes in detail16. Osmotic stress has also been shown totrigger phase separation of multivalent IDP proteins17,18. Analysis ofprotein dynamics revealed that misfolded nuclear proteins in the
nucleoplasm reversibly entered the nucleolus, especially the GC, underheat shock conditions10.
For the last 20 years, we have sought to identify key molecules
involved in neurodegenerative diseases. For example, HMGB1 was
identiﬁed by proteome analysis of polyglutamine (polyQ) disease19,
Ku70 by interactome analysis of Huntington’s disease (HD)20, and
MARCKS, SRRM2, HMGB1, and YAP by phosphoproteome analysis of
Alzheimer’s disease pathology21–24. In polyQ diseases, we searched forproteins that bind to polyglutamine tract sequences by hypothesizingthat such interacting proteins could act as disease modiﬁers25. These
analyses resulted in the identiﬁcation of polyglutamine binding protein 5 (PQBP5), along with polyglutamine binding protein 1 (PQBP1),
which was found to be an RNA transcription/splicing-related moleculethat regulates neural stem cell proliferation and neuronal synapses23–28,
as well as acting as an intracellular receptor for HIV1 and Tau, regulating inﬂammatory responses of the cGAS-STING pathway in innate
immune cells29–31. These analyses also resulted in the identiﬁcation ofpolyglutamine binding protein 3 (PQBP3), which has not yet investigated. Interestingly, most of these key molecules (e.g., MARCKS,SRRM2, YAP, and PQBP1) were IDPs.
Nucleolar proteome analysis later identiﬁed PQBP5 as a nucleolar
protein, which was designated nucleolar protein 10 (NOL10)32 containing a WD repeat33. A meta-analysis of genome-wide association
studies revealed that a single nucleotide polymorphism (SNP) in
PQBP5/NOL10 (ID: rs9287719) was a risk factor for prostate cancer34. To
date, however, gene mutations in PQBP5/NOL10 have not been
extensively analyzed in neurodegenerative or neurological diseases. In
addition, GWAS Catalog database has reported that PQBP5/NOL10 was
associated with cardiac troponin levels, arterial stiffness, waist circumference, and liver ﬁbrosis, but not with polyQ diseases (https://www.ebi.ac.uk/gwas/genes/NOL10), although it is unclear whether
46,231 human SNPs linked to PQBP5/NOL10 gene (https://www.ncbi.
nlm.nih.gov/snp/?term=NOL10) have been examined in previous
GWAS studies of polyQ diseases35–38. Expression proﬁle databases suchas Expression Atlas (https://www.ebi.ac.uk/gxa/home) have shown thatPQBP5/NOL10 is widely distributed in human/mouse neural and nonneural cells, tissues, and organs. Collectively, current lack of association at the SNP and GWAS levels does not exclude the possibility thatPQBP5 is involved in polyQ disease pathology at levels of RNA, protein,protein interaction, protein modiﬁcation, and/or protein degradation.
PQBP5 may also be an IDP whose nucleolar function is associated with
nucleolar stresses and, ultimately, with neurodegeneration including
polyQ diseases.
Intriguingly, PQBP1, 3, and 5 form a membrane-less substructurein the nucleus, with the IDP-based nuclear or cytoplasmic speckle of
PQBP1 being affected morphologically and functionally by mutant
polyQ proteins27 or by cellular stress39. In addition, SRRM2, a key IDP in
the early stage of neurodegeneration identiﬁed, as shown by comprehensive proteome analysis, forms nuclear bodies together withPQBP123, suggesting the signiﬁcance of IDP-based and membrane-lessNature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w

nuclear substructures, including the nucleolus, in neurodegeneration.
Therefore, understanding the role of PQBP5/NOL10 in the morphology
and function of the nucleolus in physiological and pathological conditions would lead to investigations of the role of the nucleolus in thepathophysiology of neurodegenerative diseases.
As the ﬁrst step of this line of research, we investigated themolecular characteristics, nucleolar sublocalization, relationship withother nucleolar proteins, and stress responses of PQBP5/NOL10. Usingsuper-resolution microscopy (SRM) and atomic force microscopy(AFM), this study found that PQBP5/NOL10 is a new type of IDP
showing a unique GC-speciﬁc distribution in the nucleolus, distinct
from that of other nucleolar proteins, and forming the skeletal structure of the nucleolus. During these investigations of the mechanisms
by which various cellular stresses affect multiple IDPs, including
PQBP5, which we selected as key molecules in neurodegeneration
based on multiple comprehensive analyses, we observed that osmotic
stress induced differential responses of IDPs, with PQBP5 being themost stable. Unexpectedly, PQBP5/NOL10 was found to act in the
nucleolus as a spatial anchor for the assembly of other nucleolar
proteins under normal conditions and during recovery from osmotic
stress. Deﬁciency of PQBP5 by knockdown resulted in the absence of
nucleoli from cells. Consistently with these analyses of the physiological function of PQBP5, pathological sequestration of PQBP5 to polyQ
aggregates in cells and mouse models, which mimic knockdown
experiments, resulted in the depletion of nucleolar PQBP5 and the loss
or abnormal structures of the nucleolus, suggesting a new aspect ofpolyQ disease pathology.

Results
PQBP5 shows a unique distribution pattern in the nucleolus
Two programs for identifying IDPs, IUPred (https://iupred2a.elte.hu/)
and RONN40, predicted that PQBP5 was an IDP containing an intrinsically disordered region (IDR) at its C-terminal end (Fig. 1a). These
programs also predicted that other nucleolar proteins, such as ﬁbrillarin and nucleolin, are IDPs (Fig. 1a). This prediction was conﬁrmed byhigh-speed AFM, which showed that PQBP5 was composed of a globular region and an unstructured dynamically mobile tail (Fig. 1b, Supplementary Movie 1). AFM showed that part of these PQBP5 molecules
were in contact with an RNA molecule, either via a relatively large
globular structure transiently formed at the proximal end of the IDR(Fig. 1b, Supplementary Movie 2) or via a small globular structuretransiently formed at the terminal end of the IDR (Supplementary
Movie 3). These molecular dynamics were illustrated schematically(Fig. 1b, schemes). The transient formation of small structures in the
IDP region occurred stochastically and independently of the presenceof RNA. These small structures could not contact RNA unless RNA waspresent nearby (Supplementary Movie 1). To address whether thetransient structure is related to interaction, we determined the numbers of times PQBP5 did or did not contact RNA in the presence orabsence of the transient structure by evaluating images taken at 1 s
intervals (Supplementary Movies 2 and 3). Fischer’s exact test suggested that the structure is related to binding (Fig. 1b, tables). These
intramolecular structures observed by AFM were well matched withthe structure of PQBP5 predicted by AlfaFold (https://alphafold.ebi.ac.
uk/) (Fig. 1c).
Morphological analysis of immunostained HeLa cells by SRM(Fig. 1d) revealed that PQBP5 formed a granular dot pattern in thecenter of the GC, as well as partially overlapping with ﬁbrillarin, amarker of DFC11 (Fig. 1e, f, g). Fibrillarin was distributed at or aroundthe FC/DFC, as reported11 (Fig. 1e). Nucleolin was present in the GC, aspreviously described10,13, but was predominantly present at the periphery of the nucleolus (Fig. 1e). By contrast, PQBP5 did not show sucha peripheral predominance, but was distributed throughout thenucleolus (Fig. 1e). The signal intensity patterns of PQBP5, nucleolin,
and ﬁbrillarin differed markedly (Fig. 1f), indicating that their2

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distributions are regulated by more complex biophysical factors.
Similar speciﬁc distributions of these three proteins have been generally observed in multiple cell types (Supplementary Fig. 1).
The 3D reconstruction of slice images obtained by SRM revealed
that PQBP5 formed a type of meshwork that could constitute theskeleton or frame of the nucleolus (Fig. 1g, Supplementary Movie 4). By
contrast, nucleolin formed peripheral condensates, and ﬁbrillarin
formed granular structures inside the nucleolus (Fig. 1g). Similar

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ﬁndings were repeatedly conﬁrmed using two SRMs from different
companies (Supplementary Figure 2, Supplementary Movies 5−7).
The electron microscopy structures corresponding to the PQBP5
granules in the GC were identiﬁed by correlative light and electron
microscopy (CLEM) (Supplementary Fig. 3). The unique distribution of
PQBP5 signals, as determined by immunocytochemistry and analyzed
by SRM, exactly matched the electron-dense meshwork structurecomposed of the small granules ﬁlling the gap space of the GC among3

Articlehttps://doi.org/10.1038/s41467-022-35602-w

Fig. 1 | PQBP5 forms the skeletal structure of the nucleolus. a Prediction of
intrinsically disordered regions (IDRs) in human PQBP5, ﬁbrillarin, and nucleolin
proteins by two independent programs. The IDR in PQBP5 was predicted at the Cterminus, whereas the IDRs in ﬁbrillarin and nucleolin were predicted at theirN-termini. b High-speed AFM reveal PQBP5 protein with a globular region and a
dynamically moving IDR tail of PQBP5 (upper panel, see Supplementary Movie 1),
PQBP5 with a small spherical domain near the globular region interacting with RNA(middle panel, see Supplementary Movie 2) or PQBP5 with a small sphericalstructure transiently formed at the IDR tail and interacting with RNA (lower panel,see Supplementary Movie 3). The relationship between PQBP5-RNA contact andtransient globular structure formation was assessed by two-sided Fisher’s exact
test. The similar observation was repeated more than three times. c Structure of
PQBP5 predicted by AlfaFold (https://alphafold.ebi.ac.uk/). d Representative superresolution microscopy (SRM) image of HeLa cells showing the spatial relationshipamong PQBP5, ﬁbrillarin, and nucleolin proteins at four nucleoli in a nucleus. DAPI

shows the area of the nucleus. The nucleolus within the white line square is further
shown in Fig. 1e, whereas the other three nucleoli are shown in Supplementary
Figure 1. The similar observation was repeated more than ten times. e Merged
image of the indicated nucleolus showing single and two-protein images. SmallPQBP5 dots were distributed homogeneously throughout the nucleolus, distinguishing PQBP5 from nucleolin and ﬁbrillarin. PQBP5 dots were in contact with
nucleolin or ﬁbrillarin dots, but did not merge with them. The similar observation
was repeated more than ten times. f Analysis of signal intensities of PQBP5,
nucleolin, and ﬁbrillarin, showing their distinct distribution patterns. g 3D image ofthe three proteins generated by Imaris (see Supplementary Movie 4 for originaldata). h Correlative light and electron microscopy (CLEM) of the three nucleolar
proteins. The most electron dense area corresponds to DFC stained with antibody
to ﬁbrillarin (red), whereas anti-PQBP5 antibody stained the middle electron dense
skeletal structure of the nucleolus (green). The similar observation was repeatedmore than ten times.

the FC/DFCs determined by electron microscopy (Fig. 1h, Supplementary Fig. 3). As expected, CLEM images of four representative
nucleoli revealed that the FC/DFCs determined by electron microscopy matched the ﬁbrillarin staining determined by SRM (Supplementary Fig. 3). CLEM also conﬁrmed that ﬁbrillarin localized the DFC
and partial overlapped with PQBP5, which was mainly localized in the
GC (Supplementary Fig. 3).
Surface plasmon resonance (SPR) spectroscopy showed a weakinteraction between PQBP5 and nucleolin but no interaction between
PQBP5 and ﬁbrillarin (Supplementary Fig. 4a). Fibrillarin, however,interacted with nucleolin (Supplementary Fig. 4a). Immunoprecipitation reconﬁrmed the similar patterns of interaction among PQBP5,
nucleolin, and ﬁbrillarin (Supplementary Fig. 4b). These results were
useful for considering the interactive relationships among PQBP5,
nucleolin, and ﬁbrillarin, together with the results of experiments
testing the effects of knockdown (KD) of one protein on anotherprotein and on co-droplet formation, as shown below.
Under normal conditions, NPM1 and Pescadillo ribosomal biogenesis factor 1 (PES1) are distributed in the GC, predominantly in the
peripheral region13,14,20. Because NPM1 has been reported to circumscribe ﬁbrillarin at the DFC as a ring, rather than being homogeneously
distributed in the GC41,42, we therefore further compared the distribution patterns of these three GC proteins, PQBP5, NPM1, and PES1,
in the nucleolus (Supplementary Fig. 5). Our results showed that the
distribution patterns of PQBP5, NPM1, and PES1 differed completely(Supplementary Fig. 5). PQBP5 was a major component of the GC, with
PES1 and NPM1 surrounding the PQBP5-containing core of thenucleolus, generating the lamellar outer shell of the GC (Supplementary Fig. 5), a ﬁnding consistent with previous reports2,10.

domain is especially critical for the nucleolar distribution of PQBP5(Supplementary Fig. 6).

IDR is essential for nucleolar distribution of PQBP5
To determine the domain or motif responsible for the nucleolar distribution of PQBP5, deletion mutants of PQBP5 were generated (Supplementary Figure 6). PQBP5 contains seven WD repeats(Supplementary Figure 6), which form a globular beta-propeller
structure or a WD repeat domain43, in agreement with the results ofhigh-speed AFM (Supplementary Movies 1–3). The deletion of WD
repeats 1 through 4 (Δ1–Δ4 mutants) did not affect the nucleolar distribution of PQBP5 (Supplementary Figure 6). PQBP5-IDR contains
coiled coil domains and a NUC 153 domain, which is homologous to ashort sequence of NUC 153 and conserved in a novel nucleolar protein
family44, (https://www.ebi.ac.uk/interpro/entry/InterPro/IPR012580/#
PUB00016366; https://www.ncbi.nlm.nih.gov/Structure/cdd/PF08159;
http://www.ebi.ac.uk/interpro/entry/pfam/PF08159) (Supplementary
Fig. 6). Partial to complete deletion of IDR irrespecive of deletion of
WD repeats (Δ5–Δ8 mutants) changed the distribution pattern of
PQBP5 from the nucleolus to the cytoplasm (Supplementary Fig. 6). A
comparison of the Δ4 and Δ5 mutants indicated that the NUC 153

Nature Communications | (2023)14:9

PQBP5 is a dominant determinant essential for nucleolus
formation
Knockdown experiments of PQBP5, nucleolin, and ﬁbrillarin were
performed to evaluate the possible role of PQBP5 as a framework fornucleolus formation (Fig. 2). Transient transfection of a pAV-U6-GFP
vector expressing shRNA for one of the three proteins (PQBP5,
nucleolin or ﬁbrillarin) (Fig. 2a) was performed to evaluate the effect ofdepletion of each of these proteins (Fig. 2b) on the distribution of theother two (Fig. 2c). Knockdown of PQBP5 markedly reduced thenucleolar concentrations of nucleolin and ﬁbrillarin and induced their
homogeneous distribution in the nucleus (Fig. 2c, upper panels), but
did not reduce the total amounts of nucleolin and ﬁbrillarin proteins
within a short period of time (Fig. 2b). Knockdown of ﬁbrillarin did notaffect the nucleolar concentrations of PQBP5 and nucleolin (Fig. 2c,
middle panels), whereas knockdown of nucleolin reduced thenucleolar concentration of ﬁbrillarin but did not affect that of PQBP5(Fig. 2c, lower panels). Original images of shRNA-mediated knockdown
are also shown (Supplementary Fig. 7).
Immunoelectron microscopy testing the effect of knockdown of
one of the three proteins on the number and shape of the nucleoli
further indicated that PQBP5 has the largest effect on nucleolus formation among the three proteins (Supplementary Fig. 8). Western
blotting showed that PQBP5, ﬁbrillarin, and nucleolin were similarly
knocked down (Supplementary Fig. 8a). A low level of expression of
PQBP5-shRNA-EGFP in cells (gold particles of anti-EGFP antibody
against shRNA-EGFP < 80/μm2 visual ﬁeld) resulted in nucleolar
deformation, whereas high levels of PQBP5-shRNA-EGFP (gold particles > 80/μm2 visual ﬁeld) mostly resulted in the absence of all nucleoli(Supplementary Fig. 8a, third row panels). The number of nucleolusnegative cells was highest in the presence of PQBP5-shRNA-EGFP
expression (Supplementary Fig. 8b).
Nucleoli in HeLa cells expressing PQBP5-shRNA have features
such as irregular shape, low electron density, and DFC disappearance(Supplementary Fig. 8c). “Abnormal cells” were therefore deﬁned ascells with nucleoli having two or more of these abnormal features. The
effect of knockdown of each protein on the percentage of abnormal
cells, irrespective of their expression levels, was therefore tested(Supplementary Fig. 8d). The frequencies of abnormal cells, as shownby the shape of the nucleoli, were higher in gold particle-positive cellswith shRNA-EGFP expression for PQBP5-KD than with shRNA-EGFP
for nucleolin- or ﬁbrillarin-KD (Supplementary Fig. 8d).
Collectively, these ﬁndings suggest that PQBP5 is the mostdominant regulator among the three IDPs (PQBP5, ﬁbrillarin, andnucleolin) (Fig. 2d), and indicate that PQBP5 is essential for nucleolus
formation.

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PQBP5 anchors and maintains the nucleolus under osmotic
stress conditions
Our preliminary experiments testing the effects of various cellularstresses on candidate IDPs, selected from our comprehensive screenings as key molecules in neurodegeneration, found that osmotic stressdifferentially modiﬁed distributions of PQBP5 and partner molecules.
In addition, because dehydration frequently occurs in elderly
people45,46 and during hyperglycemic episodes in patients with

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diabetes mellitus47–49 and because dehydration-induced delirium50,51
and diabetes melitus52 are risk factors for dementia, the function of
PQBP5 in the nucleolus was further assessed under osmotic stressconditions. HeLa cells were incubated for 20 min under hypo- or
hyper-osmolar conditions, after adjustment with sorbitol, and cultured
under normal osmotic conditions (Fig. 3a). Sequential samplings andconfocal microscopy showed that nucleolin and ﬁbrillarin, but not
PQBP5, dispersed immediately in culture medium at 220 or 500/5

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Fig. 2 | PQBP5 is essential for nucleolar assembly of ﬁbrillarin and nucleolin.
a Structure of the PQBP5, ﬁbrillarin and nucleolin knockdown vectors. The target
sequences with the loop region were subcloned between the BamHI and HindIIIsites. b Western blot analysis of HeLa cells transfected transiently with plasmids
expressing PQBP5, ﬁbrillarin, and nucleolin shRNAs. These shRNAs reduced thelevels of expression of PQBP5, ﬁbrillarin, and nucleolin to nearly 30% of the levels in
untransfected cells. The similar experiment was repeated three times.
c Immunocytochemistry showing the levels of expression of each target protein incells transfected transiently with plasmids expressing PQBP5, ﬁbrillarin, andnucleolin shRNAs. The expression of each target protein was almost completely
suppressed in transfected cells. Knockdown of PQBP5 induced dispersion ofﬁbrillarin and nucleolin. Fibrillarin knockdown did not affect the distribution of theother proteins. Nucleolin knockdown affected the distribution of ﬁbrillarin but notof PQBP5. The box plot shows median, 25–75th percentile, and whiskers representing data outside the 25–75th percentile range. Tukey’s HSD test was used for
multiple comparisons. P < 0.0001 in all comparisons by GraphPad Prism.
d Hierarchy of PQBP5, nucleolin, and ﬁbrillarin determined by knockdown
experiments.

700 mOsm (Fig. 3b). During the recovery phase, nucleolin and ﬁbrillarin promptly reassembled in an area that had retained PQBP5(Fig. 3b). Quantitative analyses of the signal intensity of nucleolin andﬁbrillarin also showed that both had dispersed to the nucleoplasm in
response to osmotic stress (Fig. 3c).
SRM yielded similar results, showing the dispersion of thenucleolar proteins under osmotic stress conditions and their reassembly in the nucleolus during recovery (Supplementary Fig. 9).
Although the signals of dispersed ﬁbrillarin appeared relatively low insingle slice images (140 nm) of SRM, the maximum intensity projection
method based on the maximum number of voxels in all projections
revealed that ﬁbrillarin dispersed under both hypo- and hyper-osmolar
conditions (Supplementary Fig. 10).

suggested that PQBP5 affected the nucleolus directly rather thanindirectly via 47S rRNA.

Relationship of nucleolar protein assembly, higher orderchromatin structure, and Pol I transcription
Nucleoli can also be evaluated by DAPI staining, which can distinguishbetween high and low-density areas of chromatin ﬁbers. We therefore
compared DAPI and nucleolar protein staining patterns in normal HeLa
cells (Supplementary Fig. 11). Nucleoli stained by antibodies to ﬁbrillarin and nucleolin tended to be located in DAPI negatives area surrounded by high DAPI signals, although such areas were not always
nucleoli and vice versa. These ﬁndings indicated that DAPI staining
could not replace nucleolar protein staining for evaluation of nucleoli.
DAPI staining also showed that knockdown of PQBP5 abrogated
the assembly of nucleolar proteins but did not alter chromatin ﬁberfree spaces for generation of nucleolus in the short term (Fig. 2).
Similar ﬁndings were observed when dispersion of nucleolar proteins
was assessed after changes in osmotic conditions (Fig. 3). Higher orderchromatin structures kept the positions for nucleolar proteins toreassemble to form nucleoli, suggesting that the order of nucleolus
formation consisted initially of rDNA exposure followed by theassembly of nucleolar proteins, within PQBP5 playing an essential rolein the latter. This hypothesis is consistent with the current view that
nucleoli are formed at nucleolus organizing regions (NORs), where
higher order chromatin structures such as chromatin ﬁbers arerelaxed, exposing rDNAs to the RNA polymerase I (Pol I) transcription
machinery53,54. Determining the chronological relationship between
higher order chromatin structure and nucleolar proteins requires
extensive investigation.
Inhibition of Pol I-driven transcription can cause re-localization of
nucleolar proteins into the nucleoplasm/cytoplasm and loss of
nucleolar integrity. Therefore, the consequences of depleting PQBP5
may be due to the loss of 47S rRNA transcription, which could be
independent from any structural role of PQBP5. Because PQBP5 binds
RNA, the effects of PQBP5 loss on 47S rRNA transcription should be
evaluated; that is, it is important to assess whether PQBP5 affects thenucleolus directly or indirectly via 47S rRNA. We therefore examined
47S rRNA, PQBP5 protein, and GAPDH protein levels (Supplementary
Fig. 12) in parallel with PQBP5 knockdown (Fig. 2). Forty-eight hours
after shRNA transfection, when the nucleolus was changed morphologically (Fig. 2), PQBP5 protein was decreased in western blot while
47S rRNA, which was evaluated by qPCR, was unchanged (Supplementary Fig. 12). At 72 h, however, both PQBP5 protein and 47S rRNA
were decreased (Supplementary Fig. 12). These results collectively

Nature Communications | (2023)14:9

LLPS of PQBP5 reconstructs the nucleolus in vitro
PQBP1, another protein that binds to polyglutamine tract sequences,
was one of the ﬁrst IDPs that showed the distribution of LLPS dropletsin the nucleoplasm outside the nucleolus27. In addition, PQBP1 formed
lamellar structure droplets with another nuclear protein, ataxin-1(Atxn1)27,55. These ﬁndings suggested the need to evaluate the biophysical features of PQBP5, nucleolin, and ﬁbrillarin in LLPS and toanalyze their differences and mutual relationships.
Because AFM revealed an interaction between PQBP5 and rRNA
(Fig. 1b) and because PQBP5-KD impairs assembly of ﬁbrillarin andnucleolin (Fig. 2), we hypothesized that the presence of rRNA would
inﬂuence droplet formation of PQBP5 or multiple IDPs mediated by
PQBP5. Therefore, droplet formation by PQBP5, nucleolin and ﬁbrillarin, each at a concentration of 250 nM, was examined in the absenceor presence of 2.5 μg/ml HeLa cell rRNA (Fig. 4a). As expected, droplet
formation by PQBP5, nucleolin, and ﬁbrillarin was markedly increasedby the addition of rRNA (Fig. 4a). In the absence of rRNA, only PQBP5
formed a few droplets, while nucleolin never and ﬁbrillarin rarely
formed droplets. The addition of rRNA enhanced PQBP5 droplet formation and increased the sizes of these droplets (Fig. 4a). The additionof rRNA also increased the number and sizes of ﬁbrillarin droplets,
although these enlarged droplets were smaller than the PQBP5 droplets (Fig. 4a). Moreover, the ﬁbrillarin droplets were unstable anddisappeared 9 h after formation (Fig. 4a). Even in the presence of rRNA,
only a very few nucleolin droplets were observed (Fig. 4a).
In the presence of HeLa cell rRNA (Fig. 4a), droplet formation was
also assessed in mixtures of two or three proteins (Fig. 4b). PQBP5 or
ﬁbrillarin alone did not enhance nucleolin droplet formation at 3 hours(Fig. 4b), whereas nucleolin cooperatively formed triple protein droplets in the presence of both PQBP5 and ﬁbrillarin (Fig. 4c). Interestingly, ﬁbrillarin formed a ring around nucleolin in vitro (Fig. 4c), thereverse of the in vivo spatial relationship, in which ﬁbrillarin forms DFC
inside nucleolin in the nucleolus. The physical characteristics of the
background ﬂuid and additional co-existing IDPs could affect droplet
formation of two IDPs by changing the Helmholtz free energy56.
Therefore, opposite relationships may occur in droplet assay bufferin vitro and nucleoplasm in vivo, including a number of other IDPs.
The resistance of each of these protein droplets to osmoticchanges was also examined (Fig. 4d). PQBP5 droplets were stable,
whereas ﬁbrillarin droplets were sensitive to osmotic changes (Fig. 4d),in agreement with the results of confocal microscopy and SRM (Fig. 3,
Supplementary Figs. 9 and 10).

The three nucleolar proteins differ in physicochemical
properties
To investigate the physicochemical features of IDPs that could explain
the relative stability of PQBP5 in comparison to ﬁbrillarin andnucleolin, we performed thermal shift assays in normal and abnormal
osmotic conditions. Thermal shift assays based on dye labeling ofhydrophobic amino acids have been used to investigate structuralchanges in proteins in response to altered temperatures. Assays using
recombinant PQBP5, nucleolin, and ﬁbrillarin proteins showed that6

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Fig. 3 | PQBP5 anchors the nucleolus under osmotic stress conditions.
a Experimental protocol for the induction of osmotic stress. b Changes in the
nucleolar distribution of nucleolar proteins during and after osmotic stress, as
shown by the relationships of PQBP5 with ﬁbrillarin and nucleolin in a single cell.
HeLa cells subjected to three types of osmotic stress showed dispersion of ﬁbrillarin and nucleolin, but retention of PQBP5, in the nucleolus. During the recovery

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phase, ﬁbrillarin and nucleolin returned to the area of PQBP5 retention.
c Quantiﬁcation of signal intensity/area (signal density) in the nucleus outside thenucleolus during and after osmotic stress, as shown by the relationships of PQBP5
with ﬁbrillarin and nucleolin. Fibrillarin and nucleolin dispersed from the nucleolus
to the nucleoplasm during stress, whereas PQBP5 did not. Data are presented asmean values ± SEM.

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nucleolin was immediately labeled with thermal shift dye at 25 °C,
whereas ﬁbrillarin was less labeled and PQBP5 was hardly labeled(Fig. 5a, left panels). The protein structures predicted by AlfaFold(https://alphafold.ebi.ac.uk/) indicated that the total length of degenerated regions was longest in nucleolin, moderate in ﬁbrillarin, andNature Communications | (2023)14:9

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shortest in PQBP5 (Fig. 5b). The other regions consisted almost completely of α-helices (Fig. 5b).
Previous studies of the effects of changing temperature from150 °C to 550 °C on multiple α-helical structures indicated that thesestructures were stable at 100 °C57. Therefore, the signal changes

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Fig. 4 | Biophysical characteristics underlying the anchor function of PQBP5.
a Puriﬁed PQBP5, nucleolin, and ﬁbrillarin proteins were ﬂuorescently labeled at
different wavelengths, and the frequency and size of droplets were investigated in
the absence or presence of ribosomal RNA. The lower graphs show quantitative
analyses of eight chips (observed area 100 μm × 100 μm). The right agar gel conﬁrms the quality of ribosomal RNA after puriﬁcation. In the absence of RNA (leftpanels and graphs), small numbers of PQBP5 droplets were observed 6 and 9 h after
mixing the ﬂuorescent protein with buffer. A few ﬁbrillarin droplets were observed
at 3 and 6 h, whereas no nucleolin droplets were detected. In the presence of RNA(right panels and graphs), PQBP5 droplets appeared immediately after mixing, with
the number peaking at 3 hours, and the sizes continuing to increase for up to 9 h.
The number and size of ﬁbrillarin droplets both peaked at 3 h, but these droplets
disappeared thereafter. A few nucleolin droplets were observed only at 3 h. Data arepresented as mean values ± SEM. b Mixtures of two nucleolar proteins under isotonic conditions. Neither PQBP5 nor ﬁbrillarin cooperatively formed droplets with
nucleolin (left and middle panels), whereas PQBP5 and ﬁbrillarin formed droplets
cooperatively (right panels). The similar experiment was repeated more than ﬁvetimes. c Formation of co-droplets by the three nucleolar proteins. Initially, PQBP5
and nucleolin surrounded ﬁbrillarin, but their lamellar relationship was reversed at
3 h. The similar experiment was repeated more than ﬁve times. d Effect of osmolarity on droplet formation by the three nucleolar proteins. PQBP5 droplets weremost resistant to changes in osmolarity. Fibrillarin droplets did not form under
hypertonic and hypotonic conditions, whereas few nucleolin droplets wereobserved under all conditions. The similar experiment was repeated more thanﬁve times.

observed from 25 °C to 100 °C in this experiment were considered toreﬂect structure changes of IDP regions (IDR). The signals of PQBP5,
nucleolin, and ﬁbrillarin that have undergone complete degeneration,
except for the α-helices, may be high at 25 °C, depending on the total
length of the degenerated regions. These signals therefore would not
be increased by increasing the temperature to 100 °C.
These assays were subsequently modiﬁed by altering the osmolarity of the reaction solution. The signals were increased under bothhypotonic and hypertonic conditions at all temperatures, with
nucleolin showing a greater increase than ﬁbrillarin (Fig. 5a, rightpanels). The variation in the ﬂuorescence of PQBP5 among isotonic,
hypotonic, and hypertonic conditions (8779.6 ﬂuorescence units) was
similar to or slightly higher than the background change of the buffer(5218.0 ﬂuorescence units), while the variation in osmotic stress was
far higher in the ﬂuorescence of nucleolin (346,818.9 ﬂuorescence
units) and ﬁbrillarin (157,697.4 ﬂuorescence units) (Fig. 5a, rightpanels), indicating that signiﬁcant structural changes were not induced
in PQBP5-IDR by hypo- or hyperosmolarity and that PQBP5 is structurally most stable among these three IDPs under osmotic stressconditions. In addition, these results indicated that PQBP5 is structurally most stable among the three IDPs under thermal stress conditions
in vitro (Fig. 5a, left panels).

Similar reductions in PQBP5, nucleolin, and ﬁbrillarin wereobserved in the nucleoli of striatal neurons of mutant Htt-KI mice
(HdhQ111 knockin mice) and of cortical neurons or Purkinje cells of
mutant Atxn1-KI mice (Sca1154Q/2Q knock-in mice) (Fig. 7a). Electron
microscopy showed that the features of the nucleoli in some of theneurons of the two KI mice were abnormal (Fig. 7b), similar to ﬁndings
in cultured PQBP5-KD cells (Supplementary Fig. 8c, d).
SPR (Supplementary Fig. 14) analyses of interactions between
polyQ disease proteins and full-length PQBP5 (FL-PQBP5) conﬁrmedthat FL-PQBP5 interacted with both normal and mutant forms of HttExon1 and Atxn1 proteins in vitro. Co-immunoprecipitation analyses
using U2OS cells transiently expressing FLAG-FL-PQBP5 and Myc-Httor Myc-Atxn1 basically supported these interactions (Supplementary
Fig. 15a, b), except for the in vivo interaction between FL-PQBP5 andnormal Htt. The latter result was due probably to the localization of FLPQBP5 in the nucleoli while normal Htt was distributed in the cytoplasm (Fig. 6). Normal Atxn1, however, co-localized with FL-PQBP5 in
the nucleoplasm (Fig. 6).
Co-immunoprecipitation analyses of polyQ disease proteins withPQBP5 deletion mutants lacking different numbers of WD repeats65,66
or the NUC 153 domain44 (Supplementary Fig. 15) suggested that the
WD repeats of PQBP5 were necessary for these interactions. Interestingly, although mutant Atxn1 had a slightly higher binding afﬁnity (Kd)to PQBP5 than normal Atxn1 (Supplementary Fig. 14), the amounts of
PQBP5 co-immunoprecipitated differed only slightly between normal
and mutant Atxn1 (Supplementary Fig. 15). On the other hand, the Kdof normal Htt-exon1 to PQBP5 was an order of magnitude higher thanthat of mutant Htt-exon1 (Supplementary Fig. 14), with the amounts of
PQBP5 co-immunoprecipitated differing markedly (Supplementary
Fig. 15). These ﬁndings suggested that sequestration of PQBP5 in
inclusion bodies composed of insoluble or less mobile mutant polyQ
disease proteins could be the cause of PQBP5 reduction in the
nucleolus. In addition, subsequent processes, such as degradation of
the complexes with mutant proteins, would contribute to the reduction of PQBP5.

PQBP5 deprivation by polyglutamine disease protein impairs
the nucleolus
PQBP5 was originally identiﬁed as a protein that bound to the polyQtract sequence associated with polyQ disease25, with the results of thisstudy showing that PQBP5 plays an essential role in nucleolar maintenance under physiological and stress conditions (Figs. 1–4). PQBP5 isessential for maintaining the structure of the nucleolus, although
PQBP5 expression in the nucleolus could be reduced following
sequestration of interacting proteins in inclusion bodies resulting fromthe generally accepted pathomechanism of polyQ diseases predictedby the “sequestration hypothesis”. The sequestered proteins becomeless mobile, as their partner disease proteins are insoluble58–62. Theseﬁndings prompted us to hypothesize that interactions between PQBP5
and polyQ disease proteins might impair the morphology of thenucleolus, which can lead ultimately to nucleolar dysfunction. To test
this hypothesis, we ﬁrst investigated the interactions between PQBP5
and polyQ disease proteins and addressed the resultant effects.
Normal and mutant Atxn1, the protein causing spinocerebellar
ataxia type 1 (SCA1)63, and Huntingtin (Htt), the protein causing Huntington’s disease (HD)64, were transiently expressed in U2OS cells(Fig. 6). Mutant Atxn1 and mutant Htt-Exon1 proteins, but not normal
Atxn1 or normal Htt-Exon1 protein, sequestered PQBP5 and dispersed
nucleolin and ﬁbrillarin (Fig. 6, left panels). Quantitative analysis conﬁrmed that PQBP5 signals were sequestered to areas containing
mutant Atxn1 or mutant Htt-Exon1 aggregates and that the nucleolar
signals of nucleolin and ﬁbrillarin were reduced (Fig. 6, right graphs).
Such tendencies were generally observed in visual ﬁelds at low magniﬁcation (Supplementary Fig. 13).

Nature Communications | (2023)14:9

Discussion
The present study was designed to reveal the physiological andpathological functions of PQBP5, a protein originally identiﬁed as aprotein binding to polyQ-tract sequences by the yeast two-hybrid
method25. We originally hypothesized in 1998 that proteins binding topolyQ sequences could be modiﬁers of polyQ diseases25. Before evaluating the pathological function of PQBP5, however, it was necessary
to characterize its biophysical properties and functions. To characterize the features of IDPs, it is ﬁrst necessary to determine theirbiophysical properties in response to altered conditions, such as heatand osmolarity. The present study showed that PQBP5 is relatively
stable in comparison with other IDPs in the nucleolus, indicating therole of PQBP5 in anchoring the nucleolus. PQBP5 knockdown reduced
the nucleolar concentrations of nucleolin and ﬁbrillarin before thedecrease of 47S rRNA, indicating that PQBP5 affected the nucleolus
9Articlehttps://doi.org/10.1038/s41467-022-35602-w

Fig. 5 | Thermal and osmotic shift assays reveal differences in the physicochemical properties of the three nucleolar proteins. a Thermal shift assay (leftpanels). Florescence signal intensities of the three nucleolar proteins at an osmolarity of 300 mOsm while changing the temperature from 25 °C to 99 °C. The frequency of exposure of hydrophobic amino acids at the outer surfaces of PQBP5,

nucleolin, and ﬁbrillarin differed markedly among the three proteins. Thermal shift
assays under multiple osmotic conditions (right panels) showing that the three
nucleolar proteins differed in their susceptibilities to changes in osmolarity.
b Structures of PQBP5, nucleolin, and ﬁbrillarin predicted by AlfaFold2.

directly rather than indirectly via 47S rRNA. We therefore investigated
the role of PQBP5 in the pathology of polyQ diseases. Consistent withthe sequestration hypothesis, we found that PQBP5 was absent frompolyQ protein inclusion bodies in polyQ diseases. These ﬁndings
suggested that conditions observed in polyQ diseases are similar to
those observed following PQBP5-KD, with depletion of PQBP5 leading
to the loss or marked deformity of the nucleolus in neurons.
The nucleolus is an essential subcellular and suborganelle
structure in eukaryotic cells and animal bodies67–70. The nucleolus is

now recognized as an LLPS-based structure1–12, with several studies
investigating the structure, component molecules, and functions of
the nucleolus71. For example, ﬁbrillarin was shown to be involved insorting rRNAs from the FC/DFC border, where rRNAs aresynthesized71,72, to the DFC11. NPM1, a component protein of the GC,
is considered critical for nucleolar structure5,12,73,74. In addition, manystudies have investigated the effects of various types of cell stresseson the morphology and function of the nucleolus14. For example, acomprehensive and quantitative proteome analysis revealed the

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localization shift of proteins between nuclear and nucleolar fractions induced by DNA damage75,76, while attention was not focusedon non-shifted proteins. In response to heat shock, Chromobox 2(CBX2), a protein component of the polycomb group complex, isconcentrated in the nucleolus; however, CBX2 is not regarded as ananchor of the nucleolus, with the phenotype considered as a protective response for CBX277. To date, however, the functional roles

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of all nucleolar components, including PQBP5 and PQBP3, have notbeen investigated completely. In addition, molecule-based responses under osmotic stress have not been determined sufﬁciently.
Therefore, some molecular mechanisms required for the maintenance of nucleoli under physiological conditions and for theassembly of multiple proteins and RNAs have not been completely
determined.
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Fig. 6 | Sequestration of PQBP5 in inclusion bodies results in the dispersion of
nucleolar proteins. Mutant Htt and Atxn1 were expressed in U2OS cells, and theireffects on PQBP5 and other nucleolar proteins were investigated. Upper panels
show sequestration of PQBP5 to cytoplasmic inclusion bodies of mutant Htt andnuclear inclusion bodies of mutant Atxn1. Expression of mutant Htt and mutant
Atxn1 resulted in the dispersion of nucleolin to the nucleoplasm. Lower panels
show the effect of PQBP5 sequestration on nucleolar assembly of ﬁbrillarin. Rightgraphs show quantitative analyses of the signal intensity of PQBP5 sequestered topolyglutamine disease protein inclusions (upper), the percentage of nucleolin
nucleolar foci-positive cells among transfected cells (middle), and the percentage
of ﬁbrillarin nucleolar foci-positive cells among transfected cells (lower). The box
plot shows median, 25–75th percentile, and whiskers representing data outside the
25–75th percentile range. Tukey’s HSD test was used for multiple comparisons.
P = 0.0002 in Atxn1-33Q vs Atxn1-86Q comparison of PQBP5 intensity in DsRedpositive area and P < 0.0001 in other comparisons by GraphPad Prism.

By contrast, nucleolar dysfunction has been implicated as apathological domain of polyQ diseases. For example, expanded CAG
repeat-containing RNAs have been found to directly interact with
nucleolin, preventing binding to the rRNA promoter and impairing
nucleolar function78. Conditional knockout of the RNA polymerase
I-speciﬁc transcription initiation factor IA (TIF-IA) has been reported tocause Huntington’s disease-like striatal degeneration79. Moreover,dysfunction of upstream binding factor-1 (UBF-1) has been reported tocontribute to HD pathology in mouse and cellular models by reducing
ribosomal DNA (rDNA) transcription80.
The present study showed that PQBP5, which was originallyidentiﬁed as a protein binding to the polyQ-tract sequence25,26, is the
key molecule that maintains and regulates nucleolar structure underboth normal and pathological conditions. Without PQBP5, othernucleolar proteins such as ﬁbrillarin and nucelolin, could not assemble
to form the nucleolus, and PQBP5 was found to function as an anchor
for the reassembly of ﬁbrillarin and nucelolin after osmotic stress. This
study also expanded knowledge about the super-resolution structure
and biophysics of PQBP5, showing that it is a self-assembling IDP that
forms a meshwork structure in the GC. Presumably, this protein could
constitute the skeleton of the nucleolus due to its relatively high biophysical stability under thermal and osmotic changes, and could
function as an anchor for the reassembly of some nucleolar proteinsafter nucleolar stress.
The present study also showed that the indispensable physiological function of PQBP5 could be hindered by its pathologicalsequestration to inclusion bodies of mutant polyQ proteins and by
resultant depletion of nucleolar PQBP5. Although PQBP5 was found tointeract with normal polyQ protein, nucleolar PQBP5 was found to be
selectively depleted in the presence of mutant polyQ proteins presumably due to immobilization to ﬁbrillary inclusion bodies60–62. Many
previous studies indicated interaction-based sequestration of physiological nuclear proteins into nuclear or cytoplasmic inclusion bodies ofmutant polyQ disease proteins; this sequestration, however, does notoccur with normal polyQ disease proteins due to the lack of sticky
immobilization, even if interactions occur81–83. Similarly to the general
concept, we found that the forced expression of mutant polyQ diseaseproteins in cultured cells and the physiological-level expression ofmutant polyQ disease proteins in KI mice leads to nucleoli withabnormal morphology, ﬁndings not observed with normal polyQ disease proteins. The sequestration of PQBP5 by mutant polyQ diseaseproteins suggested that the instability of the nucleolus due to a deﬁciency in an anchor protein could lead to neurodegeneration. Further
investigations are needed to conﬁrm this hypothesis and to reveal thedetailed mechanism underlying the association between PQBP5 andmutant polyQ diseases.
Unexpectedly, high-speed AFM showed that PQBP5 interactedwith contaminating RNA in protein samples derived from Escherichia
coli. Therefore, we evaluated the species and amount of the interacting
RNA. Agar gel electrophoresis of co-puriﬁed RNAs showed faint bands
corresponding to 23S and 16S ribosomal RNA, as well as faint low
molecular smears around 1.0 kb and below 0.5 kb (Supplementary
Fig. 16a). RT-PCR did not detect mRNA or tRNA abundantly expressedin E. coli (Supplementary Fig. 16b), indicating that the RNA molecules
would be small bacterial rRNAs such as 5S rRNA, the counterpart of
5.8S mammalian rRNA, degraded rRNA or spliced peptide fragments oftRNA (tiRNA)84,85. Based on their relative abundance, we assumed that
the RNA presumably originated from rRNA. Consistent with thishypothesis, we found that the addition of total rRNA enhanced droplet
formation of PQBP5 strongly and that of ﬁbrillarin weakly.
In these high-speed AFM experiments, the protein samples contained 2.5 ng/μl RNA (or 9.7 nmol/l based on mean size) and the PQBP5
concentration was 500 nmol/L, a ratio of about 1:50. Under these
conditions, we observed fourteen PQBP5 molecules, including two
bound and one un-bound to RNA. The dynamics of PQBP5-RNA
interactions should be further investigated, as should the identiﬁcation of the RNA species interacting with PQBP5. Determination of themechanisms by which various nucleolar proteins interact with different types of RNAs or proteins, including those that are associated withneurodegeneration, may enable in silico simulation using a supercomputer of the components and functions of nucleolus that shift
dynamically under physiological and pathological conditions.
Another issue is the relationship between proteasome-dependent
protein degradation and nucleolar protein dispersion in the nucleus
under osmotic stress conditions and in response to polyQ protein
expression. Our results showed that ﬁbrillarin is dispersed but was still
expressed, with the ﬁbrillarin signals being weaker in response to
polyQ protein expression than to osmotic stress. Hyperosmotic stresshas been reported to induce nuclear foci, including proteasomal proteins and VCP86. These ﬁndings suggested that LLPS foci function inprotein degradation, based on the correlation between proteasome
inhibitor treatment and the number/size of foci and the effect of ubiquitin inhibitor pre-treatment and formation of foci86. However, there
is no evidence showing protein degradation at LLPS foci. Conversely,
LLPS foci may be a warehouse for proteasome proteins in which they
do not function, such that some proteins may not be digested byproteasomes under osmotic stress conditions but that mutant proteins are degraded. Alternatively, these results may be due to differences in duration of the two experiments. Nucleolar proteins were
evaluated for 24 h, whereas cells were subjected to osmotic shock for
only 20 min, during which time the proteasome system was
activated86. By contrast, the effects of polyQ protein expression wereobserved for 72 h after transfection. Even if the levels of proteasome
activation were similar, proteins would be more degraded in the polyQ
expression experiments. Further studies are needed to determine themechanism underlying differences in the rates of nucleolar protein
degradation.
Consistent with ﬁndings showing that PQBP5 functions to anchor
the nucleolus to the rRNA transcription site, high-speed AFM analysis
suggested that PQBP5 interacts with cleaved rRNA, and droplet assays
indicated that rRNA promotes the self-assembly of PQBP5 and its coassembly with other nucleolar proteins. The apoptosis-antagonizing
transcription factor AATF/Che-1/TRB87–89, neuroguidin (NGDN), andPQBP5/NOL10 have been reported to form a complex for generating
the 40S ribosomal subunit90. Therefore, PQBP5-rRNA interaction could
be the basis for localizing nucleoli at NORs. Future analysis may ﬁll a
gap in knowledge of the general concept of 40S ribosome
generation69.
The mechanisms by which PQBP5 interacts with different aggregation states of Atxn1 and htt should also be analyzed. Solid-state NMR
studies of htt ﬁbrils indicate that polyQ proteins form a dense core,often excluding polyQ binding proteins such as anti-polyQ

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Fig. 7 | Effects of neurodegenerative proteins on PQBP5 and nucleolus
decomposition. a Immunohistochemistry of PQBP5 together with normal or
mutant polyglutamine disease proteins. PQBP5 was sequestered to the inclusion
bodies formed by mutant Htt in the striatal neurons of mutant Htt-KI mice (upper
left panels) and in the inclusion bodies formed by mutant Atxn1 in the corticalneurons of mutant Atxn1-KI mice (upper middle panels). In some Purkinje cells of
mutant Atxn1-KI mice, PQBP5 was sequestered to cytoplasmic inclusions (upper
right panels). In all cases, ﬁbrillarin and nucleolin lost their nucleolar assembly
activity. Fibrillarin disappeared from the nucleoli, and nucleolin was dispersed in
the nucleus. The right graphs in the left and middle columns show quantiﬁcation of
PQBP5-sequestered cells (upper graphs), cells lacking nucleolar ﬁbrillarin (middle
graphs), and cells lacking nucleolar nucleolin (lower graphs). The box plot shows
median, 25–75th percentile, and whiskers representing data outside the 25–75th

percentile range. Two-sided Student’s t-test was used for statistical examination.
P-values are as follows. % PQBP5-sequestered cells: 0.0017, % ﬁbrillarin-absent cells:
0.0053, % nucleolin-absent cells: 0.0003 in striatal neurons of Htt-KI mice at
78 weeks. % PQBP5-sequestered cells: 0.0018, % ﬁbrillarin-absent cells: 0.0095, %nucleolin-absent cells: 0.0026 in cerebral cortex neurons of Atxn1-KI mice at
32 weeks. % PQBP5-sequestered cells: 0.0011, % ﬁbrillarin-absent cells: 0.0002, %nucleolin-absent cells: 0.0006 in Purkinje cells of Atxn1-KI mice at 32 weeks. b EM
analysis of nucleoli in Purkinje cells of mutant Atxn1-KI mice and in striatal neuronsof mutant Htt-KI mice. The right bar graph shows the percentage of abnormal cells
having morphological features of abnormal nucleoli (irregular shape, low electron
density, loss of FC/DFC) in four mouse genotypes. Data are presented as meanvalues ± SEM. Tukey’s HSD test was used for multiple comparisons. P < 0.0001 incomparisons by GraphPad Prism.

antibodies91. Thus, PQBP5 may initially interact with soluble forms ofpolyQ proteins before being pulled into larger inclusions. This isconsistent with the hypothesis that LLPS-based droplets of puremutant protein mature to aggregates of ﬁbrils92, as well as with results
showing that LLPS droplets composed of multiple normal proteins,
such as PQBP1 and cGAS, remain soluble and are able to activate thecGAS-STING signaling pathway30,93. Studies are needed to determine
the mechanism by which aggregation inhibitors that prevent inclusion
formation affect the distribution of PQBP5 and other nucleolar

proteins in polyQ disease pathology. Such an approach might provide
novel ideas for future therapeutics.

Nature Communications | (2023)14:9

Methods
Plasmid construction
To construct pCMV-3Tag1A plasmids expressing full-length humanPQBP5 and PQBP5 deletion mutants, total RNA was isolated from
HEK293 cells (TaKaRa, Kusatsu, Shiga, Japan), and reverse transcribed
to cDNA. Full-length PQBP5, encoding amino acids 1-688, was13

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ampliﬁed using the primers 5′-CCCGAATTCATGCAGGTCTCCAGC-3′(forward) and 5′-GGGGGTACCTCAATGAAACGACCGTC-3′ (reverse). In
addition, sequences corresponding to amino acids 82-688, 205-688,
341-688, and 482-688 were ampliﬁed using the forward primers 5′CCCGAATTCATGACCTATCAATTAT-3′, 5′-CCCGAATTCATGCCAAGAACTCGAA-3′, 5′-CCCGAATTCATGATTCCAGTTTTGG-3′, and 5′-CCCGAATTCATGGATGATCGATTTA-3′, respectively, and the reverse primer
5′-GGGGGTACCTCAATGAAACGACCGTC-3′. The sequence corresponding to amino acids 1-481 was ampliﬁed using the primers 5′CCCGAATTCATGGATGATCGATTTA-3′ (forward) and 5′-GGGGGTACCTCAATGAAACGACCGTC-3′ (reverse), and the sequences corresponding to amino acids 1-340, 1-204, and 1-81 were ampliﬁed using
the forward primer 5′-CCCGAATTCATGCAGGTCTCCAGC-3′ and thereverse primers 5′-ATGCGGTACCTCAGTAATAGATGCCCATCTTGGG3′, 5′-ATGCGGTACCTCAGTCCCAGCACTCCACTCTA-3′, and 5′-ATGCGGTACCTCAGTCATAACATCGAACCCGAG-3′, respectively. After digestion with EcoRI and KpnI, each of these sequences was subcloned into
pCMV-3Tag-1A. In addition, the full-length human PQBP5 cDNA,
ampliﬁed as above, was subcloned into the plasmid pEGFP-N1.
To construct pET-28a plasmids expressing full-length humanPQBP5 and PQBP5 deletion mutants, sequences corresponding toamino acids 1-688, 82-688. 205-688, 341-688, and 482-688 were
ampliﬁed from HEK293 cDNA, prepared as above using the forward
primers 5′-ATGCGAATTCATGCAGGTCTCCAGCC-3′, 5′-CCCGAATTCATGCAGGTCTCCAGCC-3′, 5′-CCCGAATTCATGCCAAGAACTCGAA-3′5′-CCCGAATTAATGATTCCAGTTTTGG-3′, and 5’-CCCGAATTCATGGATGATCGATTTA-3′, respectively, and the reverse primer 5′-CATG
CGGCCGCTCAATGAAACGACCGTC-3′, The sequence corresponding
to amino acids 1-481 was ampliﬁed using the forward primer 5′-CCC
GAATTCATGCAGGTCTCCAGC-3′ and the reverse primer 5′-GGGGCG
GCCGCTCAGGTGAGAATATTA-3′. Each of these sequences was digested with EcoRI and NotI and subcloned into pET-28a.
A pET-28a plasmid expressing human ﬁbrillarin was constructedby amplifying a ﬁbrillarin sequence from HEK293 cDNA using the primers 5′-CATGCGGCCGCTCAGTTCTTCACCTTGGGGGGT-3′ (forward)
and 5′-ATGCGAATTCATGAAGCCAGGATTCAGTCCCC-3′ (reverse). A
pET-28a plasmid expressing human nucleolin was constructed byamplifying a nucleolin sequence from HEK293 cDNA using the primers
5′-CATGAATTCCTATTCAAACTTCGTCTTCTT-3′ (forward) and 5′ATGCCATATGGTGAAGCTCGCGAAGGCA-3′ (reverse). Each of thesesequences was digested with EcoRI and NdeI and subcloned into
pET-28a.
The pGEX-6P-1ATXN1-33Q/86Q plasmid was constructed bydigesting the plasmid pCMV-myc-AT1-33Q/86Q with EcoRI and NotIand subcloning the resulting Atxn1-33Q/86Q cDNA into pGEX-6P-194.
The plasmid ATXN1-33Q/86Q-DsRed was constructed by subcloning
full-length ATXN1 cDNA into the plasmid pDsRed-monomer-C1(Clontech, Mountain View, CA, USA) between the XhoI and EcoRI
sites. The Htt-17Q/103Q-DsRed plasmid was constructed by amplifying
Htt-exon 1 cDNA from pEGFP-Htt-17Q/88Q using the primers 5’-CATGAATTCTATGGCGACCCTGGAAAAG-3’ (forward) and 5’-CATGGATCCTCACGGTCGGTGCAGCGGCT-3’ (reverse) and subcloning thesequence between the EcoRI and BamHI sites of pDsRedmonomer-C194.

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imidazole, ﬁnal pH 8.0) containing 0.1 mg/ml lysozyme and proteaseinhibitor cocktail (#539134, Merck Millipore, Burlington, MA, USA).
After 30 min on ice, the cells were sonicated using an Ultrasonic Dispersion Machine UH-50 at level 6 for 10 min and centrifuged at
16,000 × g for 20 min at 4 °C. His6-tagged proteins were puriﬁed on NiNTA agarose (#30210, Qiagen, Hilden, Germany), which was washedwith wash buffer (25 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1%
Triton X-100, 20 mM imidazole, ﬁnal pH 8.0). The proteins were eluted
with elution buffer (25 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1%
Triton X-100, 250 mM imidazole, ﬁnal pH 8.0) and stored at −80 °C.
Protein samples were quantiﬁed by CBB staining and stored at −80 °C
before use. Although RNase inhibitor was not used, the protein samples included a smear of small RNAs on agar gel electrophoresis. RTPCR for three or four control genes of E. coli95 showed no evidence ofmRNA or tRNA contamination.

High-speed AFM
A glass sample stage (2 mm in diameter and 2 mm in height) with a thinmica disk 1 mm in diameter and 0.05 mm thick, bonded with epoxy,
was attached to the top of a Z scanner with drops of nail polish. New
mica surfaces were prepared by removing the top layer of mica withadhesive tape. One drop (2 μe of protein (about 3 nM) diluted withdilution buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl) was attachedto the mica surface. After incubation for 3 min, the mica surface wasrinsed with 20 μi of observation buffer to remove the suspended
sample. The sample stage was immersed in a liquid cell containing
about 60 μe of the same observation buffer (20 mM Tris-HCl, pH 7.5).
HS-AFM observation was performed in tapping mode using alaboratory-built device96,97. The custom-made short cantilever (BLAC7DS-KU4, Olympus, Tokyo, Japan) had a resonant frequency of
about 1 MHz and a quality factor of about 2 in water, as well as a spring
constant of 0.1–0.15 N m−1. The free vibration amplitude of the cantilever, A0, was set to 1–2 nm, and the set point amplitude, As, to about
0.9 × A0, with the average loss of vibration energy of the cantilever per
tap adjusted to 1–3 kBT.

Protein preparation for SPR analysis
Full-length and deletion mutant PQBP5 and polyQ proteins wereexpressed in E. coli cells (#71400, Sigma Aldrich) by transformation of
pET-28a-human PQBP5 and pET-28a-human PQBP5 deletion mutants
or by transformation of pGEX-6P-1-AT1-33Q; pGEX-6P-1-AT1-86Q;
pGEX-3X-Htt-17Q; and pGEX-3X-Htt-103Q98, as described above. E. coli
cells were collected by centrifugation, and His-tag PQBP5 fusion proteins were puriﬁed as described above. For GST-polyQ fusion proteins,the pellets were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM
NaCl, 1 mM DTT, 0.1% Triton X-100) containing 0.1 mg/ml lysozyme
and protease inhibitor cocktail (#539134, Merck Millipore). After
30 min on ice, the cells were sonicated at level 6 for 10 min and centrifuged at 16,000 × g for 20 min at 4 °C. GST fusion proteins wereadded to glutathione Sepharose 4B (#17-0756-05, GE Healthcare,
Chicago, IL, USA). After washing with wash buffer (50 mM NaH2PO4,
300 mM NaCl, 1 mM DTT), the proteins were eluted with elution buffer(50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 20 mM glutathione).

SPR analysis
Sample preparation for high-speed AFM and Droplet assays
Rosetta™ 2(DE3) Singles™ Competent E. coli cells (#71400, Sigma
Aldrich, St. Louis, MO, USA) were transformed individually with pET28a-human ﬁbrillarin, pET-28a-human nucleolin, and pET-28a-humanPQBP5. The bacteria were grown to an OD600 of 0.5 and incubated with
1 mM IPTG for 18 hours at 16 °C to induce expression of the fusion
protein, followed by the centrifugation of 800 ml of each bacterial
culture at 3, 000 × g for 20 min at 4 °C. To purify His-tag fusion proteins, the pellets were resuspended in lysis buffer (25 mM Tris-HCl,
500 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 10 mM

Nature Communications | (2023)14:9

SPR analysis was performed using a Biacore T100 instrument (Cytiva,
Marlborough, MA, USA) at 25 °C. Interactions among the proteinsPQBP5, ﬁbrillarin, and nucleolin were assessed by immobilizing 5 ng/mlof each protein dissolved in 10 mM sodium acetate on a CM5 sensor
chip until the immobilized proteins had gained 200 resonance units(RU). Multiple concentrations of analytes (0, 5, 10, 20, 30, 40, and
50 nM) were injected for 180 s at a rate of 10 μl/min. Sample binding
and dissociation were assessed in HBS-EP+ buffer for 300 s. Except forthe binding of PQBP5 as analyte and nucleolin as immobilized protein,
all binding experiments were performed in His-tagged protein elution
14

Article
buffer without imidazole (25 mM Tris-HCl, 500 mM NaCl, 10% glycerol,
1% Triton X-100, ﬁnal pH 8.0). The sensor chip was regenerated byinjection of 10 mM glycine-HCl (pH 2.1) for 60 sec at a rate of 30 μl/min
to perform kinetic analysis.
To assess the binding of polyglutamine disease proteins (GSTAtxn1-33Q, GST-Atxn1-86Q) to PQBP5 or its deletion mutants, 5 ng/mlof the Atxn1 proteins in 10 mM sodium acetate were immobilized on a
CM5 sensor chip until the immobilized proteins had gained 11,000 RU.
Multiple concentrations of analytes (0, 12.5, 25, 50, 100, and 200 nM)
were injected for 180 s at a rate of 10 μl/min. Sample binding anddissociation were assessed in HBS-EP+ buffer for 300 s. The sensor chip
was regenerated as described above.
To determine the binding of polyglutamine disease proteins (GSTHtt-20Q, GST-Htt-110Q) to PQBP5 or its deletion mutants, 5 ng/ml Httproteins in 10 mM sodium acetate solution were immobilized to 1100
RU on a CM5 sensor chip. Multiple concentrations of analytes (0, 12.5,
25, 50, 100, and 200 nM) were injected for 180 s at a rate of 20 μl/min.
Sample binding and dissociation were assessed in HBS-EP+ buffer for
300 s, and the sensor chip was regenerated as described above.

Immunoprecipitation
HeLa cells (RCB0007), a kind gift from Naoyuki Kataoka that waspurchased from RIKEN BRC Cell Bank (Tsukuba, Japan), were cultured
with Dulbecco’s Modiﬁed Eagle’s Medium–high glucose (D5796 medium, Sigma Aldrich, St. Louis, MO, USA) with 10% Fetal Bovine Serum(#10270106, Gibco, MA, USA), transfected with the plasmids of pCMV3Tag1A-Full-length PQBP5 or PQBP5 deletion mutants and pCMV-mycAT1-33Q/86Q or pCMV-myc-Htt-Exon1-17Q/103Q, and harvested 48 hlater. U2OS cells, a generous gift from Professor Yoshio Miki (Tokyo
Medical and Dental University) that was purchased from ATCC (Manassas, VA, USA), were cultured in Dulbecco’s Modiﬁed Eagle’s
Medium–high glucose (D5796 medium, Sigma Aldrich, St. Louis, MO,
USA) with 10% Fetal Bovine Serum (#10270106, Gibco, MA, USA),transfected with the plasmids of pCMV-3Tag1A-Full-length PQBP5,
pCMV-myc-AT1-33Q/86Q or pCMV-myc-Htt-Exon1-17Q/103Q, andharvested 48 h later.
The cells were lysed with TNE buffer (10 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing proteaseinhibitor cocktail (#539134, Merck Millipore). The lysates were rotatedfor 60 min at 4 °C and centrifuged at 12,000 × g for 1 min at 4 °C. Eachsupernatant was incubated with a 50% slurry of Protein-G Sepharose
beads (17061801, GE Healthcare, Chicago, IL, USA) for 2 h at 4 °C, followed by centrifugation at 2000 × g for 2 min at 4 °C. The supernatants
were incubated with 2 μp antibody for 5 h at 4 °C with rotation, followed by the addition of 40 μl Protein-G Sepharose and rotation for 2 h
at 4 °C. The beads were washed three times with TNE buffer, and 30 μl
sample buffer (125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 5% (v/v) 2mercaptoethanol, 10% (v/v) glycerol, and 0.0025% (w/v) bromophenol
blue) were added to each sample. The samples were boiled at 95 °C for
10 min, followed by SDS-PAGE and transfer to Immobilon-P polyvinylidene diﬂuoride membranes (Merck Millipore). The membranes
were incubated with various primary antibodies, including rabbit antiFLAG antibody (#F7425, Sigma); mouse anti-Myc antibody (#M047-3,
MBL, Aichi, Japan); rabbit anti-ﬁbrillarin antibody (1:1000 #ab166630,
Abcam, Cambridge, UK); rabbit anti-nol10 antibody (1:5000 #ab181161
Abcam); and mouse anti-nucleolin antibody (1:5000 #ab13541 Abcam).

Super-resolution microscopy
HeLa cells were ﬁxed in 4% formaldehyde for 15 min at RT, permeatedwith 0.1% Triton X-100, and blocked by incubation in blocking buffer(50 mM Tris-HCl pH 6.8, 150 mM NaCl, and 0.1% Tween-20 in MQ)
containing 1 mg/ml BSA and 300 mM glycine for 60 min at RT. The
samples were incubated with mouse anti-ﬁbrillarin (1:100, #ab4566,
Abcam), mouse anti-NPM1 (1:5000, #ab10530, Abcam), rat anti-PES1
antibody (1:2000, #252849, Abcam), or rabbit anti-NOL10/PQBP5Nature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w(1:150 #ab181161 Abcam) antibody for 60 min at RT, followed byincubation for 60 min at RT with the secondary antibodies Alexa Fluor
555-conjugated anti-mouse IgG (1:1000, #A31570, Molecular Probes,
Eugene, OR, USA), donkey anti-mouse IgG (H + L) Highly CrossAdsorbed Alexa Fluor 647 (1:1000, #31571, Thermo Fisher Scientiﬁc,Waltham, MA, USA), donkey anti-rat IgG (H + L) Highly Cross-Adsorbed
Alexa Fluor 488 (1:1000, #21208, Thermo Fisher Scientiﬁc, Alexa Fluor
488-conjugated anti-rabbit IgG (1:1000, #A21206, Molecular Probes)
or donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Alexa Fluor
568 (1:1000, #10042, Thermo Fisher Scientiﬁc). For nucleolin staining,
the samples were incubated with 10% normal goat serum for 60 min,followed by incubation for 60 min at RT with mouse anti-nucleolin
antibody (1:2000 #ab13541 Abcam) that had been labeled with the
ZenonTM Alexa Fluor 647 Mouse IgG1 labeling kit (#z25008, Thermo
Fisher Scientiﬁc). The samples were ﬁxed with 4% formaldehyde for
15 min at RT, and the 3D images were acquired by SRM, using an Elyra 7(Carl Zeiss Co., Ltd., Oberkochen, Germany), LSM 980 with Airyscan2(Carl Zeiss Co., Ltd), or IX83 (Olympus, Tokyo, Japan) microscope
equipped with a CSU-W1 SoRa Spinning Disk Confocal (Yokogawa
Electric Corporation, Tokyo, Japan), an ORCA Flash4.0 V3 digital CMOS
camera (Hamamatsu, Shizuoka, Japan), and a UPLXAPO60XO objective lens (Olympus, Tokyo, Japan).

Correlative light and electron microscopy
HeLa cells ﬁxed in 4% formaldehyde for 15 min at RT were incubatedwith antibodies to nucleolin; nol10; and ﬁbrillarin, as described above
for immunocytochemistry. The cells were visualized using an LSM 980
with an Airyscan2 super-resolution microscope (Carl Zeiss, Co., Ltd),ﬁxed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4 °C for 2 h,and incubated with 1% osmium tetroxide at 4 °C for 2 h. The cells were
dehydrated with a graded series of ethanol, and embedded in epon at
60 °C for 48 h and at 120 °C for 24 h. Ultrathin sections (80 nm) wereprepared with an ultramicrotome (US6, Leica, Wetzlar, Germany),
incubated with uranyl acetate and lead citrate, and viewed under ascanning electron microscope (JSM-7900F, JEOL, Tokyo, Japan).

shRNA-expressing plasmids
The shRNA-expressing plasmids for PQBP5 (SH837805), nucleolin(SH843928), ﬁbrillarin (SH827603) and their scramble shRNA
controls were generated by Vigene Biosciences (Rockville, MD,
USA). Brieﬂy, the shRNA sequences for PQBP5 (NM_024894),
nucleolin (NM_005381), and ﬁbrillarin (NM_001436) consisted of
5′-GAAGTCCGTTCATTTCCAGGATTCTTCAAGAGAGAATCCTGGAAATGAACGGACTTTTTTT-3′, 5′- GAAGAAGAGGACCAACATCATTCCTTCAAGAGAGGAATGATGTTGGTCCTCTTCTTTTTTT-3′ and 5′-GA
AACAGAAAGCAGCTCCTGAAGCTTCAAGAGAGCTTCAGGAGCTGC
TTTCTGTTTTTTTT-3′, respectively. Each of these sequences anda scramble control shRNA sequence were cloned into the plasmid
pAV-U6-GFP between the BamHI and HindIII sites at the 3′ terminal of the human U6 promoter.

Western blot
Forty-eight hours after transfection of shRNA plasmids, cells were
harvested and incubated for 15 min at 4 °C in lysis buffer (10 mM TrisHCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40) with proteaseinhibitor cocktail (#539134, 1:200 dilution, Calbiochem, San Diego, CA,
USA). After centrifugation at 10,000 × g for 10 min, the supernatants
were mixed with an equal volume of sample buffer (125 mM Tris-HCl(pH 6.8), 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol,and 0.0025% (w/v) bromophenol blue), separated by SDS-PAGE, andtransferred to Immobilon-P polyvinylidene diﬂuoride membranes(Merck Millipore) by the semi-dry method. The membranes were
blocked by incubation in 5% milk in TBST (10 mM Tris/HCl (pH 8.0),
150 mM NaCl, 0.05% Tween-20), and incubated overnight at 4 °C withrabbit anti-ﬁbrillarin (1:1000, #ab166630); mouse anti-C23 (1:5000,
15Article#sc-8031, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-nol10(1:5000 #ab181161, Abcam); rabbit anti-GFP (1:6000, #8334, Santa Cruz
Biotechnology), or mouse anti-GAPDH (1:6000, #MAB374, Merck
Millipore) primary antibodies, each diluted in Can Get Signal solution(Toyobo, Osaka, Japan). The membranes were subsequently incubatedwith HRP-linked anti-rabbit IgG (1:3000, #NA934, GE Healthcare) or
HRP-linked anti-mouse IgG (1:3000, #NA931, GE Healthcare) secondary antibodies for 1 h at room temperature. Proteins were detectedwith ECL Prime Western Blotting Detection Reagent (RPN2232, GE
Healthcare) and a luminescent image analyzer (ImageQuant LAS 500,
GE Healthcare). Uncropped and unprocessed scans of the blots areincluded in Source Data ﬁle.

Immunoelectron microscopy
Hela cells were transfected with 2.5 μg each of the plasmids pAV-U6GFP-nucleolin shRNA (SH843928), pAV-U6-GFP-ﬁbrillarin shRNA(SH827603), and pAV-U6-GFP-PQBP5 shRNA (SH837805), each prepared by Vigene Biosciences, using Lipofectamine 2000 transfection
reagent (Thermo Fisher Scientiﬁc, #11668019). After 48 h, the cells
were ﬁxed with ﬁxation buffer (4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer) for 15 min at 25 °C and incubatedin blocking buffer (50 mM NH4Cl, 0.1% Saponin, 1% BSA) for 1 hour at
25 °C. The cells were stained with rabbit anti-GFP antibody (1:10000,#sc-8334, Santa Cruz Biotechnology) for 30 min at 25 °C, and incubated with Nanogold®-IgG goat anti-rabbit IgG (H + L) (1:200, #2003,
Nanoprobes, Yaphank, NY, USA) for 2 h at 25 °C. Nanogold signals wereenhanced with GoldEnhanceTM EM (#2113, Nanoprobes) for 5 min at
25 °C. After ﬁxation in 1% glutaraldehyde in 0.2 M HEPES, the sections
were post-ﬁxed with 1% OsO4 for 30 min at 4 °C and dehydrated
through a graded ethanol series. The samples were incubated twicewith propylene oxide for 15 min each, once with a 1:1 mixture of propylene oxide and epon for 4 h at 25 °C, once with a 1:3 mixture of
propylene oxide and epon overnight at 25 °C, and embedded in pureepon for 3 days. Ultrathin sections (80 nm) were prepared with aultramicrotome (UC7, Leica, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The sections were observed using atransmission electron microscope (model 1400 plus, JEOL Ltd).

Osmotic stress and Immunocytochemistry
HeLa cells were incubated in hypotonic buffer (D1152 medium, Sigma),isotonic buffer (D1152 medium with 44 mM NaHCO3), hypertonic
buffer (D1152 medium with 44 mM NaHCO3 and 0.2 M sorbitol (#19114735, Fujiﬁlm, Osaka, Japan), or super-hypertonic buffer (D1152
medium with 44 mM NaHCO3 and 0.4 M sorbitol (#191-14735, Fujiﬁlm).
The cells were subsequently incubated in isotonic buffer (D1152 medium with 44 mM NaHCO3) for 0, 10, 30, 180, 720, or 1440 min at 37 °C.
Following cell ﬁxation in 4% formaldehyde for 15 min at RT, the cells
were washed three times in PBS, permeated with 0.1% Triton X-100,
and blocked with blocking buffer (50 mM Tris-HCl pH 6.8, 150 mM
NaCl, and 0.1% Tween-20 in MQ) containing 1 mg/ml BSA and 300 mM
glycine for 60 min at RT. The samples were subsequently incubatedwith both mouse anti-ﬁbrillarin (1:100, #ab4566, Abcam) or rabbit antinol10 (1:150 #ab181161, Abcam) for 60 min at RT followed by incubation with Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000, #A21206,
Molecular Probes) and Alexa Fluor 555-conjugated anti-mouse IgG(1:1000, #A31570, Molecular Probes) secondary antibodies for 60 min
at RT. For nucleolin staining, the cells were incubated with 10% normal
goat serum for 60 min, followed by incubation at RT for 60 min withmouse anti-nucleolin antibody (1:2000, #ab13541, Abcam) that had
been labeled with a ZenonTM Alexa Fluor 647 Mouse IgG1 labeling kit(#z25008, Thermo Fisher Scientiﬁc) and ﬁxation with 4% formaldehyde for 15 min at RT.
For the immunocytochemistry by super-resolution microscopy
and correlative light and electron microscopy, HeLa cells were cultured with Dulbecco’s Modiﬁed Eagle’s Medium – high glucose
Nature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w(D5796 medium, Sigma Aldrich, St. Louis, MO, USA) with 10% Fetal
Bovine Serum (#10270106, Gibco, MA, USA) for 24 h at 37 °C before
ﬁxation. For knockdown experiment, HeLa cells were transfectedwith the shRNA-expressing plasmids for PQBP5 (SH837805),
nucleolin (SH843928), ﬁbrillarin (SH827603) and their scramble
shRNA controls. Forty-eight hours after transfection, cells were ﬁxed
with 4% formaldehyde.
For immunocytochemistry by confocal microscopy, U2OS cells
were transfected with the plasmids of ATXN1-33Q/86Q-DsRed or Htt17Q/103Q-DsRed. After 72 h, cells were ﬁxed with 4% formaldehyde.

Analysis of 47 S rRNA expression after PQBP5 knockdown
HeLa cells were transfected with 3 μg of pAV-U6-GFP-scrambled shRNA
or pAV-U6-GFP-PQBP5 shRNA plasmid using Lipofectamine 2000
transfection reagent (Thermo Fisher Scientiﬁc, #11668019). The cells
were harvested at 48 or 72 h after transfection, and divided for western
blot, RT-PCR and quantitative RT-PCR analyses. For western blot analysis, cells were lysed with TNE buffer (10 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1% NP-40) containing protease inhibitor
cocktail (#539134, 1:200 dilution, Calbiochem, San Diego, CA, USA) for
15 min at 4 °C. After centrifugation at 10,000 g for 10 min), thesupernatants were mixed with an equal volume of sample buffer(125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol,
10% (v/v) glycerol, and 0.0025% (w/v) bromophenol blue), separated
by SDS-PAGE, and transferred to Immobilon-P polyvinylidene diﬂuoride membranes (Merck Millipore) by the semi-dry method. The
membranes were incubated with rabbit anti-Nol10 antibody (1:5000#ab181161, Abcam) overnight at 4 °C, followed by incubation with HRPlinked anti-rabbit IgG (1:3000, #NA934, GE Healthcare) for 1 h at room
temperature. For RT-PCR analysis, total RNA was isolated from thesamples with RNeasy mini kits (74106, Qiagen, Limburg, Netherlands).
During RNA isolation, DNA was digested on-column using DNase I toeliminate genomic DNA contamination. Reverse transcription was
performed using the SuperScript VILO cDNA Synthesis kit (11754-250,
Invitrogen, Carlsbad, CA, USA), and PCR was performed with the T100
Thermal Cycler (Bio-Rad, Hercules, CA, USA) using TaKaRa Ex Taq(#RR001A, TaKaRa, Shiga, Japan). The PCR ampliﬁcation protocol
consisted of an initial denaturation for 2 min at 95 °C, followed by 20
cycles of denaturation for 25 s at 95 °C, annealing for 30 sec at 55 °C,
and extension for 60 s at 72 °C. After mixing with loading buffer(#9157, TaKaRa), the samples were separated by 2% agarose gel electrophoresis in 1 × TAE buffer (0.04 M Tris–acetate, 1 mM EDTA). Bandintensity on western blots and agarose gels were analyzed using ImageJ software. For quantitative RT-PCR, analyses were performed with the7500 Real-Time PCR System (Applied Biosystems, Foster City, CA,
USA) using the Thunderbird SYBR Green (QPS-201, TOYOBO, Osaka,
Japan) and assessed by the standard curve method. The PCR conditions for ampliﬁcation were 95 °C for 10 min for enzyme activation,
95 °C for 15 s for denaturation, and 60 °C for 1 min for extension (40cycles).
The PCR primer sequences were:
Human 47S rRNA:99
forward primer: 5′-GCTGACACGCTGTCCTCTG-3′
reverse primer: 5′-ACGCGCGAGAGAACAGCAG-3
Human GAPDH:100
forward primer: 5′- GGTGGTCTCCTCTGACTTCAACA-3′
reverse primer: 5′- GTTGCTGTAGCCAAATTCGTTGT-3

Ribosomal RNA preparation for Droplet assay
Total RNA was isolated from HeLa cells with RNeasy mini kits (74106,
Qiagen), including on-column digestion of DNA with DNase I to eliminate genomic DNA contamination. The total RNAs were hybridized
with the single-stranded DNA probes speciﬁc for ribosomal RNAs (NEB
#7405, New England BioLabs, Ipswich, MA, USA); residual nonhybridized single-strand RNAs were degraded using RNase A
16

Article(#EN0531, Thermo Fisher Scientiﬁc), and the hybridized DNA probes
were degraded by NEBNext DNase I (NEB #7405, New England BioLabs). Ribosomal RNAs were recovered by NEBNext RNA Sample
puriﬁcation beads (NEB #7405, New England BioLabs) and storedat −80 °C.

Droplet assay
AlexaFluor 488, AlexaFluor 647, and AlexaFluor 555 were conjugatedto PQBP5, ﬁbrillarin, and nucleolin proteins, respectively, using the#A30006, #A30009, and #A30007 AlexaFluor Labeling kits (Thermo
Fisher Scientiﬁc), respectively. The reaction buffer was removed bydialysis against the low osmolarity buffer (20 mM Tris-HCl, pH 7.5,
25 mM NaCl, and 1 mM DTT) for droplet formation. All protein concentrations were adjusted to 0.25 μM in hypotonic (20 mM Tris-HCl,
pH 7.5, 125 mM NaCl, 1 mM DTT); isotonic (20 mM Tris-HCl, pH 7.5,
125 mM NaCl, 40 mM NaHCO3, 1 mM DTT); hypertonic (20 mM TrisHCl, pH 7.5, 125 mM NaCl, 40 mM NaHCO3, 0.2 M sorbitol, 1 mM DTT);
and superhypertonic (20 mM Tris, pH 7.5, 125 mM NaCl, 40 mM
NaHCO3, 0.4 M sorbitol, 1 mM DTT) solutions. Droplet formation on
silicone wells (#GBL665108-25EA, Grace BioLabs, Bend, OR, USA) in theabsence or presence of 2.5 μg/ml ribosomal RNA was assessed byincubation for 0, 3, 6, and 9 hours at 25 °C, and observed by confocal
microscopy (FV1200, Olympus).

Thermal and osmolar shift assay
PQBP5, nucleolin, and ﬁbrillarin His-tag fusion proteins were generated as described above, adjusted to 0.3 μM, and dialyzed against to
20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT using Slide-A-Lyzer
MINI Dialysis Units (#69570, Thermo Fisher Scientiﬁc). Each well of a
PCR plate (MicroAmp™ Fast Optical 96-Well Reaction Plate, #4346907,
Live Technologies, Carlsbad, CA, USA) contained 10 μl of a fusion
protein, 2.5 μl of Protein Thermal Shift dye (#4462263, Thermo Fisher
Scientiﬁc) and 7.5 μl of osmotic buffer, resulting in a ﬁnal volume of 20
μl. Four types of osmotic buffer were used: hypotonic (246.5 mM
NaCl); isotonic (353 mM NaCl); hypertonic (620 mM NaCl); andsuperhypertonic (886.5 mM NaCl). The plates were sealed with
MicroAmp Optical Adhesive Film, and the temperature was increased
from 25 °C to 99 °C at a rate of 0.4 °C per minute, during which time
the ﬂuorescence in each well was monitored. The results were analyzed
using Protein Thermal Shift™ Software v1.4 (#4466037, Thermo Fisher
Scientiﬁc).

Mice
Mutant Ataxin-1 knock-in mice (Sca1154Q/2Q mice) were a generous giftfrom Prof. Huda Y. Zoghbi (Baylor College of Medicine, Houston, TX,USA)101, and mutant Huntingtin knock-in mice (HdhQ111 mice) were agenerous gift from Prof. Marcy MacDonald (Massachusetts General
Hospital, Harvard Medical School, Boston, MA, USA)102. C57BL/6J (BL/6)
mice purchased from Sankyo Labo Service Corporation (Tokyo, Japan)
were used for breeding. Non-transgenic sibling mice were used ascontrols. All animal experiments were performed in accordance withAnimal Research: Reporting in vivo Experiments (ARRIVE) guidelines
and were approved by the Committees on Gene Recombination
Experiments and Animal Experiments of Tokyo Medical and Dental
University (G2018-082C3 and A2021-211A).

Immunohistochemistry
Mouse brains were ﬁxed in 4% paraformaldehyde for 16 h, embedded
in parafﬁn, and sectioned at a thickness of 5 μ5. The sections were deparafﬁnized in xylene and re-hydrated, followed by antigen retrieval by
microwaving in 0.01 M citrate buffer (pH 6.0) at 120 °C for 15 min. After
permeation with PBS containing 0.5% Triton X-100, the sections wereincubated with blocking solution (10% normal donkey serum in PBS)
for 60 min at room temperature, following by incubation for 24 hours
at 25 °C with mouse anti-Htt (1:100, MAB5374, Sigma Aldrich), mouse

Nature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w

anti-Atxn1 (1:100, MAB5374, Sigma Aldrich), mouse anti-ﬁbrillarin(1:100, #ab4566, Abcam), rabbit anti-nol10 (1:5000, #ab181161,
Abcam), and mouse anti-nucleolin (1:5000, #ab13541, Abcam) primary
antibodies. After washing three times in PBS at room temperature, thesections were incubated at room temperature for 1 h with Alexa Fluor
488-conjugated anti-rabbit IgG (1:1000, A21206, Molecular Probes)
and Alexa Fluor 555-conjugated anti-mouse IgG (1:1000, #A31570,
Molecular Probes) secondary antibodies.

Evaluation of RNA interacting with PQBP5 in AFM
RNAs contamination of protein samples was evaluated at multiple
steps during puriﬁcation of His-Tag PQBP5 protein for AFM. BrieﬂyRNAs extracted from intermediate and ﬁnal protein samples using
RNeasy mini kits (74106, Qiagen) were loaded onto agar gels. To
eliminate genomic DNA contamination, DNA was digested on-column
digestion using DNase I during RNA isolation.

RT-PCR
RNA was isolated from Rosetta™ 2(DE3) Singles™ Competent E. coli
cells (#71400, Sigma Aldrich) or His-PQBP5 protein samples using
RNeasy mini kits (74106, Qiagen). During this process, DNA wasdigested on-column with DNase I to eliminate genomic DNA contamination. Reverse transcription was performed using SuperScript
VILO cDNA Synthesis kits (11754-250, Invitrogen), and PCR was performed on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, U.S.A.) using
TaKaRa Ex Taq (#RR001A, TaKaRa). The endogenous controls wereidnT, hcaT, and cysG for mRNA, and leuT, serU, serW, and proK fortRNA. After mixed with loading buffer (#9157, TaKaRa), the samples
were separated by 1% agarose gel electrophoresis in 1 × TAE buffer(0.04 M Tris–acetate, 1 mM EDTA).
The primer sequences were idnT, 5′-CTGTTTAGCGAAGAGGAGATGC-3′ (forward) and 5′-ACAAACGGCGGCGATAGC-3′ (reverse);
hcaT, 5′-GCTGCTCGGCTTTCTCATCC-3′ (forward) and 5′-CCAACCACGCAGACCAACC-3′ (reverse); cysG, 5′-TTGTCGGCGGTGGTGATGTC-3′(forward) and 5′-ATGCGGTGAACTGTGGAATAAACG-3′ (reverse); leuT,
5′-GCGAAGGTGGCGGAATTGGTAGAC-3′ (forward) and 5′-TGGTGCG
AGGGGGGGGACTTGAAC-3′ (reverse); serU, 5’-GGAGAGATGCCGGAG
CGGCTGAAC-3′ (forward) and 5′-TGGCGGAGAGAGGGGGATTTGAAC3′ (reverse); serW, 5’-GGTGAGGTGTCCGAGTGGCTGAAGT-3′ (forward)
and 5′-GGCGGTGAGGGGGGGATTCGAAG-3′ (reverse); and proK, 5’CGGTGATTGGCGCAGCCTGGTAGC-3′ (forward) and 5′-GAGGATTCGAACCTCCGACCCCTT-3′ (reverse).
The ampliﬁcation conditions consisted of an initial incubation at
55 °C for 10 min, followed by denaturation at 95 °C for 2 min, 45 cycles
of denaturation at 95 °C for 10 s, and annealing and extension at 68 °C
for 60 s.

Conﬁrmation of SNP/GWAS data and polyQ disease association
44,993 SNPs related to PQBP5 were conﬁrmed using NCBI SNP
database (dbSNP: https://www.ncbi.nlm.nih.gov/snp/). To ﬁnd theirassociation with polyQ diseases, research papers including genomewide association studies of polyQ diseases were searched using
Pubmed (https://pubmed.ncbi.nlm.nih.gov/) with keywords “PolyQ
and SNP”, “Polyglutamine and SNP”, “CAG repeat and SNP”, “PolyQ
and GWAS”, “Polyglutamine and GWAS” or “CAG repeat and GWAS”,
and 140 research papers were collected. SNP or GWAS data attachedto the papers were examined by custom code for relevance to44,993 SNPs.

Statistics
Statistical analyses for biological experiments were performed using
Graphpad Prism 8 or Microsoft Excel for Microsoft 365.
Biological data following a normal distribution are presented as
the mean ± SEM, with Tukey’s HSD test utilized for multiple groupcomparisons.
17Article
The distribution of observed data was depicted with box plots, withthe data also plotted as dots. Box plots show the medians, quartiles, andwhiskers, which represent data outside the 25th–75th percentile range.

https://doi.org/10.1038/s41467-022-35602-w
14.
15.

EthicsThis study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of theJapanese Government and the National Institutes of Health. All
experiments were approved by The Committees for Gene Recombination Experiments and Animal Experiments of the Tokyo Medical and
Dental University (G2018-082C3 and A2021-211A).

18.

Reporting summary

19.

16.
17.

Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
20.

Data availability
SNPs of PQBP5 were collected NCBI SNP database (https://www.ncbi.
nlm.nih.gov/snp/). Search results of SNP/GWAS data and polyQ
disease-associated research papers are available at https://doi.org/10.
5281/zenodo.7389755. All other data generated or analyzed during thisstudy are included in this article. Source data are provided withthis paper.

21.

22.

Code availability

23.

A custom code for conﬁrmation of SNP/GWAS data and polyQ diseaseassociation are available at https://doi.org/10.5281/zenodo.7389755103.

24.

References
1.

2.
3.
4.

5.

6.

7.

8.

9.
10.
11.

12.

13.

Brangwynne, C. P., Mitchison, T. J. & Hyman, A. A. Active liquid-likebehavior of nucleoli determines their size and shape in Xenopus
laevis oocytes. Proc. Natl Acad. Sci. USA. 108, 4334–4339 (2011).
Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell
physiology and disease. Science 357, eaaf4382 (2017).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecularcondensates: organizers of cellular biochemistry. Nat. Rev. Mol.
Cell Biol. 18, 285–298 (2017).
Mitrea, D. M. et al. Self-interaction of NPM1 modulates multiple
mechanisms of liquid-liquid phase separation. Nat. Commun. 9,
1–13 (2018).
Mitrea, D. M. et al. Nucleophosmin integrates within the nucleolus
via multi-modal interactions with proteins displaying R-rich linear
motifs and rRNA. elife 5, 1–33 (2016).
Falahati, H. & Wieschaus, E. Independent active and thermodynamic processes govern the nucleolus assembly in vivo. Proc.
Natl Acad. Sci. USA 114, 1335–1340 (2017).
Weber, S. C. & Brangwynne, C. P. Inverse size scaling of thenucleolus by a concentration-dependent phase transition. Curr.
Biol. 25, 641–646 (2015).
Caragine, C. M., Haley, S. C. & Zidovska, A. Nucleolar dynamics andinteractions with nucleoplasm in living cells. elife 8, 1–21 (2019).
Frottin, F. et al. The nucleolus functions as a phase-separated protein quality control compartment. Science 365, 342–347 (2019).
Yao, R. W. et al. Nascent Pre-rRNA sorting via phase separation
drives the assembly of dense ﬁbrillar components in the humannucleolus. Mol. Cell 76, 767–783 (2019).
Ferrolino, M. C., Mitrea, D. M., Michael, J. R. & Kriwacki, R. W.
Compositional adaptability in NPM1-SURF6 scaffolding networks
enabled by dynamic switching of phase separation mechanisms.
Nat. Commun. 9, 5064 (2018).
Biggiogera, M. et al. Nucleolar distribution of proteins B23 andnuclolin in mouse preimplantation embryos as visualized byimmunoelectron microscopy. Development 110, 1263–1270 (1990).

Nature Communications | (2023)14:9

25.

26.

27.
28.

29.
30.

31.

32.

33.

34.

35.
36.

37.

Boulon, S., Westman, B. J., Hutten, S., Boisvert, F. M. & Lamond, A.
I. The nucleolus under stress. Mol. Cell 40, 216–227 (2010).
Delpire, E., Duchêne, C., Goessens, G. & Gilles, R. Effects ofosmotic shocks on the ultrastructure of different tissues and cell
types. Exp. Cell Res. 160, 106–116 (1985).
Cioce, M., Boulon, S., Matera, A. G. & Lamond, A. I. UV-induced
fragmentation of Cajal bodies. J. Cell Biol. 175, 401–413 (2006).
Jalihal, A. P. et al. Multivalent proteins rapidly and reversibly
phase-separate upon osmotic cell volume change. Mol. Cell 79,
978–990.e5 (2020).
Majumder, S. & Jain, A. Osmotic stress triggers phase separation.
Mol. Cell 79, 876–877 (2020).
Qi, M. L. et al. Proteome analysis of soluble nuclear proteins
reveals that HMGB1/2 suppress genotoxic stress in polyglutamine
diseases. Nat. Cell Biol. 9, 402–414 (2007).
Enokido, Y. et al. Mutant huntingtin impairs Ku70-mediated DNA
repair. J. Cell Biol. 189, 425–443 (2010).
Tagawa, K. et al. Comprehensive phosphoproteome analysis
unravels the core signaling network that initiates the earliest
synapse pathology in preclinical Alzheimer’s disease brain. Hum.
Mol. Genet. 24, 540–558 (2015).
Fujita, K. et al. HMGB1, a pathogenic molecule that induces neurite
degeneration via TLR4-MARCKS, is a potential therapeutic target
for Alzheimer’s disease. Sci. Rep. 6, 1–5 (2016).
Tanaka, H. et al. The intellectual disability gene PQBP1 rescues Alzheimer’s disease pathology. Mol. Psychiatry 23, 2090–2110 (2018).
Tanaka, H. et al. YAP-dependent necrosis occurs in early stages of
Alzheimer’s disease and regulates mouse model pathology. Nat.
Commun. 11, 1–22 (2020).
Imafuku, I. et al. Polar amino acid-rich sequences bind to polyglutamine tracts. Biochem. Biophys. Res. Commun. 253,
16–20 (1998).
Waragai, M. et al. PQBP-1, a novel polyglutamine tract-binding
protein, inhibits transcription activation by Brn-2 and affects cell
survival. Hum. Mol. Genet. 8, 977–987 (1999).
Okazawa, H. et al. Interaction between mutant ataxin-1 and PQBP-1
affects transcription and cell death. Neuron 34, 701–713 (2002).
Ito, H. et al. In utero gene therapy rescues microcephaly caused by
Pqbp1-hypofunction in neural stem progenitor cells. Mol. Psychiatry 20, 459–471 (2015).
Yoh, S. M. et al. PQBP1 is a proximal sensor of the cGAS-dependent
innate response to HIV-1. Cell 161, 1293–1305 (2015).
Jin, M. et al. Tau activates microglia via the PQBP1-cGAS-STING
pathway to promote brain inﬂammation. Nat. Commun. 12,
1–5 (2021).
Yoh, S. M. et al. Recognition of HIV-1 capsid by PQBP1 licenses aninnate immune sensing of nascent HIV-1 DNA. Mol. Cell 82,
2871–2884.e6 (2022).
Ahmad, Y., Boisvert, F. M., Gregor, P., Cobley, A. & Lamond, A. I.
NOPdb: nucleolar proteome database − 2008 Update. Nucleic
Acids Res. 37, 181–184 (2009).
Wada, K. et al. Dynamics of WD-repeat containing proteins in
SSU processome components. Biochem. Cell Biol. 92, 191–199(2014).
Olama, A. A. Al et al. A meta-analysis of 87,040 individuals identiﬁes 23 new susceptibility loci for prostate cancer. Nat. Genet. 46,
1103–1109 (2014).
GeM-HD Consortium. Identiﬁcation of genetic factors that modify
clinical onset of Huntington’s disease. Cell 162, 516–526 (2015).
Bettencourt, C. et al. DNA repair pathways underlie a common
genetic mechanism modulating onset in polyglutamine diseases.
Ann. Neurol. 79, 983–990 (2016).
Akçimen, F. et al. Genome-wide association study identiﬁes
genetic factors that modify age at onset in Machado-Joseph disease. Aging 12, 4742–4756 (2020).
18

Article
38.

39.

40.

41.

42.

43.

44.

45.
46.

47.

48.

49.
50.
51.

52.

53.
54.
55.

56.

57.

58.
59.

Lee, J.-M., MacDonald, M. E. & Gusella, J. F. Inherited HTT CAG
repeat length does not have a major impact on Huntington disease
duration. Am. J. Hum. Genet. 109, 1338–1340 (2022).
Kunde, S. A. et al. The X-chromosome-linked intellectual disability
protein PQBP1 is a component of neuronal RNA granules andregulates the appearance of stress granules. Hum. Mol. Genet. 20,
4916–4931 (2011).
Yang, Z. R., Thomson, R., McNeil, P. & Esnouf, R. M. RONN: the biobasis function neural network technique applied to the detectionof natively disordered regions in proteins. Bioinformatics 21,
3369–3376 (2005).
Holmberg Olausson, K., Elsir, T., Moazemi Goudarzi, K., Nistér, M.
& Lindström, M. S. NPM1 histone chaperone is upregulated inglioblastoma to promote cell survival and maintain nucleolar
shape. Sci. Rep. 5, 1–15 (2015).
Hayes, M. H., Peuchen, E. H., Dovichi, N. J. & Weeks, D. L. Dual roles
for ATP in the regulation of phase separated protein aggregates in
Xenopus oocyte nucleoli. elife 7, e35224 (2018).
Sprague, E. R., Redd, M. J., Johnson, A. D. & Wolberger, C.
Structure of the C-terminal domain of Tup1, a corepressor oftranscription in yeast. EMBO J. 19, 3016–3027 (2000).
Staub, E., Fizev, P., Rosenthal, A. & Hinzmann, B. Insights into theevolution of the nucleolus by an analysis of its protein domain
repertoire. BioEssays 26, 567–581 (2004).
Hess, T. [Thirst deﬁciency in old age]. Dtsch. Med. Wochenschr.
112, 1668–1669 (1987).
Andreucci, V. E., Russo, D., Cianciaruso, B. & Andreucci, M. Somesodium, potassium and water changes in the elderly and theirtreatment. Nephrol. Dial. Transplant. 11, 9–17 (1996).
Arnaud, P., Paille, M., Paille, J. F., Rollet, J. & Plauchu, M. [Comacaused by hyperosmolarity in the diabetic patient. Physiopathology, diagnostic and therapeutic deductions]. Presse Med. 79,
949–952 (1971).
Jackson, W. P. A critical appraisal of mechanisms concerned in
non-ketotic hyperosmolar diabetic coma. Postgrad. Med. J. 47,
399–402 (1971).
Foster, D. W. Insulin deﬁciency and hyperosmolar coma. Adv.
Intern. Med. 19, 159–173 (1974).
Inouye, S. K. Delirium in hospitalized older patients. Clin. Geriatr.
Med. 14, 745–764 (1998).
Davis, D. H. J. et al. Delirium is a strong risk factor for dementia in
the oldest-old: a population-based cohort study. Brain 135,
2809–2816 (2012).
Biessels, G. J. & Despa, F. Cognitive decline and dementia in diabetes mellitus: mechanisms and clinical implications. Nat. Rev.
Endocrinol. 14, 591–604 (2018).
Li, G. & Reinberg, D. Chromatin higher-order structures and gene
regulation. Curr. Opin. Genet. Dev. 21, 175–186 (2011).
Schöfer, C. & Weipoltshammer, K. Nucleolus and chromatin.
Histochem. Cell Biol. 150, 209–225 (2018).
Okazawa, H., Sudol, M. & Rich, T. PQBP-1 (Np/PQ): a polyglutamine
tract-binding and nuclear inclusion-forming protein. Brain Res.
Bull. 56, 273–280 (2001).
Kamimura, Y. R. & Kanai, M. Chemical insights into liquid-liquid
phase separation in molecular biology. Bull. Chem. Soc. Jpn. 94,
1045–1058 (2021).
Paschek, D., Gnanakaran, S. & Garcia, A. E. Simulations of thepressure and temperature unfolding of an α-helical peptide. Proc.
Natl Acad. Sci. USA 102, 6765–6770 (2005).
Perutz, M. F. Glutamine repeats and neurodegenerative diseases:
Molecular aspects. Trends Biochem. Sci. 24, 58–63 (1999).
Wanker, E. E. Protein aggregation in Huntington’s and Parkinson’s
disease: Implications for therapy. Mol. Med. Today 6,
387–391 (2000).

Nature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w
60.

61.

62.

63.
64.

65.

66.

67.
68.

69.
70.
71.

72.

73.

74.
75.

76.

77.

78.

79.

80.

81.
82.

Chai, Y., Shao, J., Miller, V. M., Williams, A. & Paulson, H. L. Live-cell
imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model ofpathogenesis. Proc. Natl Acad. Sci. USA 99, 9310–9315 (2002).
Stenoien, D. L., Mielke, M. & Mancini, M. A. Intranuclear ataxin1
inclusions contain both fast- and slow-exchanging components.
Nat. Cell Biol. 4, 806–810 (2002).
Kim, S., Nollen, E. A. A., Kitagawa, K., Bindokas, V. P. & Morimoto, R.
I. Polyglutamine protein aggregates are dynamic. Nat. Cell Biol. 4,
826–830 (2002).
Orr, H. T. et al. Expansion of an unstable trinucleotide CAG repeatin spinocerebellar ataxia type 1. Nat. Genet. 4, 221–226 (1993).
MacDonald, M. E. et al. A novel gene containing a trinucleotide
repeat that is expanded and unstable on Huntington’s disease
chromosomes. Cell 72, 971–983 (1993).
Neer, E. J., Schmidt, C. J., Nambudripad, R. & Smith, T. F. The
ancient regulatory-protein family of WD-repeat proteins. Nature
371, 297–300 (1994).
Smith, T. F., Gaitatzes, C., Saxena, K. & Neer, E. J. The WD repeat: acommon architecture for diverse functions. Trends Biochem. Sci.
24, 181–185 (1999).
Pederson, T. The plurifunctional nucleolus. Nucleic Acids Res. 26,
3871–3876 (1998).
Boisvert, F. M., van Koningsbruggen, S., Navascués, J. & Lamond,
A. I. The multifunctional nucleolus. Nat. Rev. Mol. Cell Biol. 8,
574–585 (2007).
Lafontaine, D. L. J. Noncoding RNAs in eukaryotic ribosome biogenesis and function. Nat. Struct. Mol. Biol. 22, 11–19 (2015).
Iarovaia, O. V. et al. Nucleolus: a central hub for nuclear functions.
Trends Cell Biol. 29, 647–659 (2019).
Lafontaine, D. L. J., Riback, J. A., Bascetin, R. & Brangwynne, C. P.
The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol.
Cell Biol. 22, 165–182 (2021).
Lafontaine, D. L. J. Birth of nucleolar compartments: phaseseparation-driven ribosomal RNA sorting and processing. Mol.
Cell 76, 694–696 (2019).
Amin, M. A., Matsunaga, S., Uchiyama, S. & Fukui, K. Depletion of
nucleophosmin leads to distortion of nucleolar and nuclear
structures in HeLa cells. Biochem. J. 415, 345–351 (2008).
Grisendi, S., Mecucci, C., Falini, B. & Pandolﬁ, P. P. Nucleophosmin
and cancer. Nat. Rev. Cancer 6, 493–505 (2006).
Boisvert, F. M. & Lamond, A. I. p53-Dependent subcellular proteome localization following DNA damage. Proteomics 10,4087–4097 (2010).
Boisvert, F. M., Lam, Y. W., Lamont, D. & Lamond, A. I. A quantitative proteomics analysis of subcellular proteome localizationand changes induced by DNA damage. Mol. Cell. Proteom. 9,457–470 (2010).
Azkanaz, M. et al. Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock. elife 8,
1–27 (2019).
Tsoi, H., Lau, T. C. K., Tsang, S. Y., Lau, K. F. & Chan, H. Y. E. CAG
expansion induces nucleolar stress in polyglutamine diseases.
Proc. Natl Acad. Sci. USA 109, 13428–13433 (2012).
Kreiner, G. et al. A neuroprotective phase precedes striatal
degeneration upon nucleolar stress. Cell Death Differ. 20,
1455–1464 (2013).
Lee, J. et al. Dysregulation of upstream binding factor-1 acetylation
at K352 is linked to impaired ribosomal DNA transcription in
Huntington’s disease. Cell Death Differ. 18, 1726–1735 (2011).
Zoghbi, H. Y. & Orr, H. T. Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci. 23, 217–247 (2000).
Orr, H. T. Beyond the Qs in the polyglutamine diseases. Genes
Dev. 15, 925–932 (2001).

19

Article
83.

Okazawa, H. Polyglutamine diseases: a transcription disorder?
Cell. Mol. Life Sci. 60, 1427–1439 (2003).
84. Yamasaki, S., Ivanov, P., Hu, G. F. & Anderson, P. Angiogenin
cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42 (2009).
85. Haussecker, D. et al. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16, 673–695 (2010).
86. Yasuda, S. et al. Stress- and ubiquitylation-dependent phaseseparation of the proteasome. Nature 578, 296–300 (2020).
87. Page, G., Lödige, I., Kögel, D. & Scheidtmann, K. H. AATF, a noveltranscription factor that interacts with Dlk/ZIP kinase and interferes with apoptosis. FEBS Lett. 462, 187–191 (1999).
88. Fanciulli, M. et al. Identiﬁcation of a novel partner of RNA polymerase II subunit 11, Che‐1, which interacts with and affects thegrowth suppression function of Rb. FASEB J. 14, 904–912(2000).
89. Lindfors, K. et al. Identiﬁcation of novel transcription factor-likegene from human intestinal cells. Biochem. Biophys. Res. Commun. 276, 660–666 (2000).
90. Bammert, L., Jonas, S., Ungricht, R. & Kutay, U. Human AATF/Che-1
forms a nucleolar protein complex with NGDN and NOL10
required for 40S ribosomal subunit synthesis. Nucleic Acids Res.
44, 9803–9820 (2016).
91. Lin, H. K. et al. Fibril polymorphism affects immobilized nonamyloid ﬂanking domains of huntingtin exon1 rather than its
polyglutamine core. Nat. Commun. 8, 1–12 (2017).
92. Ray, S. et al. α-Synuclein aggregation nucleates throughliquid–liquid phase separation. Nat. Chem. 12, 705–716(2020).
93. Du, M. & Chen, Z. J. DNA-induced liquid phase condensation ofcGAS activates innate immune signaling. Science 709,
704–709 (2018).
94. Fujita, K. et al. A functional deﬁciency of TERA/VCP/p97 contributes to impaired DNA repair in multiple polyglutamine diseases. Nat. Commun. 4, 1816 (2013).
95. Zhou, K. et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by
quantitative PCR. BMC Mol. Biol. 12, 18 (2011).
96. Ando, T. et al. A high-speed atomic force microscope for studying
biological macromolecules. Proc. Natl Acad. Sci. USA. 98, 12468
LP–12412472 (2001).
97. Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force
microscopy for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83, 337–437 (2008).
98. Imamura, T. et al. Identiﬁcation of hepta-histidine as a candidate
drug for Huntington’s disease by in silico-in vitro- in vivointegrated screens of chemical libraries. Sci. Rep. 6, 1–5(2016).
99. Juli, G. et al. Depletion of ribosomal protein S19 causes a reductionof rRNA synthesis. Sci. Rep. 6, 35026 (2016).
100. Dai, W., Toro, A., Dierschke, S. K. & Dennis, M. D. High-fat diet/palmitate-induced ER stress promotes protein O-GlcNAcylation in
retina and retinal muller cells. Diabetes 67, 607-P (2018).
101. Watase, K. et al. A long CAG repeat in the mouse Sca1 locus
replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration. Neuron 34,
905–919 (2002).
102. Wheeler, V. C. et al. Length-dependent gametic CAG repeat
instability in the Huntington’s disease knock-in mouse. Hum. Mol.
Genet. 8, 115–122 (1999).

Nature Communications | (2023)14:9

https://doi.org/10.1038/s41467-022-35602-w
103. Homma, H. & Okazawa, H. PQBP5/NOL10 maintains and anchorsthe. nucleolus under physiological and osmotic stress conditions.
Zenodo https://doi.org/10.5281/zenodo.7389755 (2022).

Acknowledgements
The authors thank Carl Zeiss Co., Ltd and Yokogawa Electric Corporation
for their generous support regarding SRM. The authors also thank Dr.
Xuemei Zhang (Tokyo Medical and Dental University) for mouse andhistology experiments, Dr. Teikichi Ikura (Ochanomizu University) for
advice on protein puriﬁcation, and Drs. Noriki Katayama and Kazuma
Tatsumi (Kanazawa University) for AFM. This work was supported by
grants to H.O., including a Grant-in-Aid for Scientiﬁc Research on
Innovative Areas (Foundation of Synapse and Neurocircuit Pathology,
22110001/ 22110002) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (MEXT), and a Grant-in-Aid for Scientiﬁc Research A (16H02655, 19H01042, 22H00464) from the JapaneseSociety for the Promotion of Science (JSPS).

Author contributions
X.J., H.T., M.J., K.F., K.U., and N.K.: acquisition and analysis of data, anddrafting of the manuscript. M.I. and H.Y.: acquisition of data, H.H. and
T.A.: analysis of data and drafting of the manuscript. H.O.: conception
and design of the study, obtaining funding, and drafting the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains
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Hitoshi Okazawa.
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