BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
A ailable online 19 Ap il 2024
1874-9399/© 2024 The Au ho . Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Re iew
The nucleolus: Coo dina ing s ess esponse and genomic s abili y
Ka iuska Gonz´
alez-A zola
*
Cen o Andaluz de Biología Molecula y Medicina Regene a i a (CABIMER), Uni e sidad de Se illa, Consejo Supe io de In es igaciones Cien í icas, Jun a de Andalucía,
Uni e sidad Pablo de Ola ide, 41092 Se ille, Spain
Depa amen o de Bioquímica Vege al y Biología Molecula , Uni e sidad de Se illa, 41012 Se ille, Spain
ARTICLE INFO
Keywo ds:
Cellula s ess coo dina ion
Nucleola s ess
Nucleolus
DNA ansc ip ion
Ribosome biogenesis
ABSTRACT
The pe cep ion ha he nucleoli a e me ely he o ganelles whe e ibosome biogenesis occu s is challenged. Only
a ound 30 % o nucleola p o eins a e solely in ol ed in p oducing ibosomes. Ins ead, he nucleolus plays a
c i ical ole in con olling p o ein a icking du ing s ess and, acco ding o i s dynamic na u e, unde goes
con inuous p o ein exchange wi h nucleoplasm unde a ious cellula s esso s. Hence, he concep o nucleola
s ess has e ol ed as cellula insul s ha dis up he s uc u e and unc ion o he nucleolus. Conside ing he
eme ging ole o his o ganelle in DNA epai and he ac ha DNAs a e he mos agile genomic loci, he apies
a ge ing he nucleoli a e inc easingly being de eloped. Besides, d ugs ha a ge ibosome syn hesis and induce
nucleola s ess can be used in cance he apy. In con as , agen s ha egula e nucleola ac i i y may be a
po en ial ea men o neu odegene a ion caused by abno mal p o ein accumula ion in he nucleolus. He e, I
Abb e ia ions: 3D FISH, idimensional luo escence in si u hyb idiza ion; A-bodies, amyloid bodies; APE1, apu inic/apy imidinic endonuclease 1; ARF, al e -
na i e eading ame; ATM, a axia elangiec asia mu a ed; ATR, a axia elangiec asia and Rad3- ela ed p o ein; BAX, BCL2-associa ed x p o ein; BER, base excision
epai ; BLM, Bloom’s synd ome p o ein; BRCA1, b eas cance ype 1 suscep ibili y p o ein; CDK, cyclin-dependen kinase; CKII, casein kinase II; CLEM, co ela i e
ligh and elec on mic oscopy; CRISPR/Cas9, clus e ed egula ly in e spaced sho palind omic epea s/caspase9; CRM1, ch omosome egion main enance 1; C BP,
ca boxy- e minal binding p o ein; C IP, C BP in e ac ing p o ein; DDR, DNA damage esponse; diRNA, damage-induced small RNA; DJ, dis al junc ion; DKC1,
dyske in pseudou idine syn hase 1; DNA-PKcs, a ca aly ic subuni o DNA-dependen p o ein kinase; DRB, 5,6-dichlo o-1-β-D- ibo u anosylbenzimidazole; DSB,
double-s and b eak; E2F1, E2F ansc ip ion ac o 1; EM, elec on mic oscopy; ETS, ex e nal ansc ibed space ; FBL, ib illa in; GAR, Gly-A g- ich domain;
GLTSCR2, glioma umo - supp esso candida e egion gene 2; GNL2, guanine nucleo ide binding p o ein-like 2; GNL3, guanine nucleo ide binding p o ein-like 3;
H3K27me3, his one H3 Lys27 ime hyla ion; H3K4me3, his one H3K4 ime hyla ion; H3K9me2/3, his one H3 Lys9 di/ ime hyla ion; H4K20me2/3, his one H4
Lys20 di/ ime hyla ion; HMG, high-mobili y g oup; HMGB1, high mobili y g oup box 1; HP1
α
/β, he e och oma in p o ein 1
α
/β; HR, homologous ecombina ion;
HS-AFM, high-speed a omic o ce mic oscopy; IDP, in insically diso de ed p o ein; IDR, in insically diso de ed egion; IGS, in e genic space ; IGS16RNA, lncRNA a
16 kb downs eam o he DNA TSS; IGS22RNA, lncRNA a 22 kb downs eam o he DNA TSS; IGS28RNA, lncRNA a 28 kb downs eam o he DNA TSS; INB,
in anucleola body; I-Ppol, Physa um polycephalum endonuclease I; ITS, in e nal ansc ibed space ; K- ich, Lys- ich; lncRNA, long non-coding RNA; L- RNA, la ge
subuni RNA; MDC1, media o o DNA damage checkpoin p o ein 1; MKI67, ma ke o p oli e a ion Ki-67; MLO, memb ane-less o ganelle; MRE11, meio ic
ecombina ion 11 homolog 1; MRN, MRE11, RAD50 and NBS1 complex; NAC, NAM/ATAF/CUC ansc ip ion ac o amily; NAD, nucleola associa ed domain;
NBS1, Nijmegen b eakage synd ome p o ein 1; NCL, nucleolin; nDDR, nucleola DNA damage esponse; NER, nucleo ide excision epai ; NHEJ, non-homologous end
joining; NHP2L1, ch omosome p o ein 2-like 1; NOL10, nucleola p o ein 10; NoLS, nucleola localiza ion sequence; NoRC, nucleola emodeling complex; NPM,
nucleophosmin; NuRD, nucleosome emodeling and deace yla ion; PAPAS, p omo e and p e- RNA an isense; PARP1, Poly ADP- ibose polyme ase 1; PCNA,
p oli e a ing cell nuclea an igen; PJ, p oximal junc ion; PML, p omyelocy ic leukemia p o ein; POL-I/II/III, RNA polyme ase-I/II/III; p e- RNA, ibosomal RNA
p ecu so ; RAD50, adia ion sensi i e p o ein 50; RAD51/52, adia ion sensi i e p o ein 51/52; RBD, RNA binding domain; RBP, RNA binding p o ein; DNA, i-
bosomal DNA; R-DPR, R- ich dipep ide- epea p o ein; RIEP, ibosomal IGS encoded p o ein; RNF8, RNF8: ing inge p o ein 8; RP, ibosomal p o ein; RPA194, RNA
polyme ase-I subuni A; RPA2, eplica ion p o ein A2; RPF2, ibosome p oduc ion ac o 2; RPL, ibosomal p o ein la ge; RPS, ibosomal p o ein small; RPSA, i-
bosomal p o ein SA; R- ich, a ginine- ich; RNA, ibosomal RNA; RRP1A, ibosomal RNA p ocessing p o ein 1 homolog A; RRS1, ibosome biogenesis egula o y
p o ein homolog; SL1, p omo e selec i i y ac o ; snoRNA, small nucleola RNA; snoRNP, small nucleola ibonucleop o ein; snRNP, small nuclea ibonucleo-
p o eins; SRM, supe - esolu ion mic oscopy; S- RNA, small subuni RNA; SUMO 1/2/3, small ubiqui in like modi ie 1/2/3; SURF6, su ei 6; TAF, TBP-associa ed
ac o s; TBA, h ombin-binding ap ame ; TBP, TATA-binding p o ein; TCOF1, T eache Collins-F ancesche i synd ome; TFIIIB, ansc ip ion ac o -IIIB; TIF-IA,
ansc ip ion ini ia ion ac o -IA; TIP5, TTF-1-in e ac ing p o ein-5; TOPO I/II, ype I/II opoisome ase; TSS, ansc ip ion s a si e; TTF1, ansc ip ion e mina ion
ac o 1; UBF, ups eam binding ac o ; WRN, We ne synd ome helicase; XRCC1, X- ay c oss complemen ing p o ein 1; XRCC4, X- ay c oss complemen ing p o ein 4;
ZTF-7, zinc inge pu a i e ansc ip ion ac o amily 7.
* A enida Am´
e ico Vespucio 24, 41092 Se ille, Spain.
E-mail add ess: [email p o ec ed].
Con en s lis s a ailable a ScienceDi ec
BBA - Gene Regula o y Mechanisms
jou nal homepage: www.else ie .com/loca e/bbag m
h ps://doi.o g/10.1016/j.bbag m.2024.195029
Recei ed 15 No embe 2023; Recei ed in e ised o m 25 Ma ch 2024; Accep ed 12 Ap il 2024
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
2
explo e he oles o nucleoli beyond hei ibosomal unc ions, highligh ing he ac o s igge ing nucleola s ess
and hei impac on genomic s abili y.
1. In oduc ion
Floa ing in he ch oma in solu ion, nucleoli a e he mos p ominen
and isible s uc u es wi hin he nucleus o euka yo ic cells. They a e
memb ane-less o ganelles (MLOs), as a e nuclea speckles, Cajal bodies,
his one locus bodies, p omyelocy ic leukemia p o ein (PML) nuclea
bodies, he e och oma in, he nuclea po e complex, among o he as-
semblies exis ing in cell nuclei ha con ibu e o compa men aliza ion
o speci ic biological unc ions [1–3].
Fo many yea s, he sole unc ion o he nucleolus was hough o be
a ac o y o ibosomal RNA ( RNA) syn hesis and assembly o ibosomal
subuni s [4]. Ne e heless, his o ganelle has gained a en ion o e he
las wo decades as a cen al hub o p o ein a icking con ol o c i ical
cellula p ocesses, e.g., cell cycle p og ession [5], umo igenesis [6], and
he cellula esponse o a la ge numbe o s esso s [7], including DNA
damage [8].
Nucleoli exhibi a highly dynamic s uc u e ha is disassembled and
eassembled in each cell cycle [9]. Such a dynamic beha io is behind i s
la ely asc ibed ole in he coo dina ion o cellula esponse o inju ies.
No in ain, di e en ypes o cellula s ess ul si ua ions a e o en
accompanied by d ama ic changes in he o ganiza ion and composi ion
o he nucleolus [7]. Beyond he ac ha nucleoli ha e been conside ed
no el playe s in sensing and managing he cellula s ess esponse, he
concep o ‘nucleola s ess’ has been inc easingly employed in ecen
yea s. This e m is now used o e e o abno mali ies in he s uc u e
and unc ion o he nucleolus caused by s ess ul e en s ha impai he
ibosome biogenesis unc ion and, consequen ly, a ec cellula ho-
meos asis [10].
Conside ing he g owing ole o he nucleolus in DNA epai and ha
ibosomal DNA ( DNA) genes, which eside in nucleoli, a e especially
uns able egions, nucleola s ess has been linked o se e al pa hologies,
anging om cance and neu odegene a i e diseases o o e all aging
[11]. Consequen ly, he apies a ge ing nucleoli a e e iginously
eme ging. In his e iew, I e isi he oles o nucleoli beyond hei i-
bosomal unc ions, emphasizing he ac o s ha igge nucleola s ess
and hei e ec s on gene s abili y egula ion.
2. Nucleola s uc u e and unc ions
Nucleoli encompass DNA, di e se ypes o RNAs, and o e 300
p o eins [4]. These compa men s we e ini ially desc ibed as he si es o
ibosome biogenesis, implica ing he ansc ip ion o DNA genes, RNA
ma u a ion, and ibosomal subuni assembly [4]. P o eins and RNAs
in ol ed in ibosome syn hesis a e highly concen a ed in he nucleolus,
c ea ing a unique compa men o dis inc body wi hou a delimi ing
memb ane. The high concen a ion o RNAs comes om he an-
sc ip ion o he ibosomal genes, and hese genes comp ise abou 60 % o
o al cellula ansc ip ion in euca yo ic cells, gua an eeing ha he
ansla ional machine y mee s he p o ein syn hesis demand [12]. To
inc ease RNA syn hesis and sus ain he high demand o ibosomes,
euka yo ic cell genomes keep a high copy numbe o hei RNA genes,
a ying om 100 o 10,000 ac oss species [13]. E en hough cells
possess an ele a ed copy numbe o DNA genes, only 50 % o hem a e
ansc ibed; e en me abolically ac i e cells wi h a high demand o i-
bosomes keep a po ion o hei DNA silenced, which has led some
esea che s o conside hese genomic si es as senso s o DNA damage
[14–16]. DNA me hyla ion o cy osine esidues in CpG dinucleo ides is
he p incipal epigene ic mechanism o silencing o DNA genes [13,17].
In addi ion, ep essi e his one ma ks a e o en ound in he p omo e
egions o silenced DNA genes. These ma ks include his one H3 Lys27
ime hyla ion (H3K27me3), H3K9me3, as well as deace yla ed his one
H4 Lys16 (H4K16) and H4K20 [18,19]. On he con a y, ac i e DNA
genes a e en iched in ac i e his one ma ks, i.e., H3K4 ime hyla ion
(H3K4me3), H3 and H4 ace yla ion, and he his one a ian H2A.Z [19].
Nucleoli o m a ound ansc ip ionally ac i e epea s o andemly
epea ed DNAs genes, a.k.a. nucleola o ganize egions (NORs Fig. 1)
[20,21], esul ing in a subnuclea compa men ha concen a es he
ansc ip ion and p ocessing machine y esponsible o RNA syn hesis,
p ocessing, and p e- ibosome subuni assembly [22]. In humans, he
DNA gene copies a e posi ioned on he sho a m o he ac ocen ic
ch omosomes (13, 14, 15, 21, and 22), be ween elome ic and cen o-
me ic he e och oma in, and dis ibu ed o e he i e ch omosomes [21]
(Fig. 1). DNA gene a ay a NORs is lanked by wo sequences named
dis al and p oximal junc ions (DJ and PJ, espec i ely, Fig. 1) ha a e
conse ed among he abo e ch omosomes [23]. NORs can exis in ei he
ac i e o silen s a es. A ac i e NORs, he DNA copies a e ex ensi ely
complexed o he main DNA ansc ip ion ac o named ups eam
binding ac o (UBF), a high-mobili y g oup (HMG) box con aining
p o ein ha s imula es RNA syn hesis. Thus, DNA genes emain unde -
condensed. NORs a e isible as seconda y cons ic ions on ch omosomes
[24] (Fig. 1). On he con a y, ansc ip ionally silen o inac i e NORs
a e no bound o UBF and, hus, en i ely condensed [25].
2.1. The nucleolus as a mul ilaye biomolecula condensa e: he nucleola
sub-s uc u e
G owing e idence suppo s ha he nucleolus ep esen s a biomol-
ecula condensa e, a e m inc easingly used o desc ibe MLOs, whose
o ma ion is d i en by e e sible liquid-liquid phase sepa a ion (LLPS)
[26,27] simila o oil-in-wa e emulsions. Thus, nucleoli a e dynamic
assemblies wi h liquid-like p ope ies whose endency o use explains
hei b oad size dis ibu ion [26]. The usion o nucleoli is a highly
ac i e and dynamic p ocess [28]. Du ing in e phase, he numbe o
nucleoli g adually dec eases as hey use o associa e wi h one ano he ,
causing he emaining nucleoli o become la ge and dense [28].
Consequen ly, one o wo nucleoli a e ypically obse ed du ing his
pe iod. Howe e , a he beginning o cell di ision (p ophase), he
nucleolus begins o dissocia e, and hen nucleoli eappea a he end o
mi osis ( elophase) [28]. The maximum numbe o nucleoli pe nucleus
depends on he maximum numbe o NORs p esen in a genome, which
is 10 in human cells [29].
The nucleolus’s highly dynamic a chi ec u e, which is disassembled
and eassembled in each cell cycle a ound ac i e NORs, esul s om he
immiscibili y o hei di e en luid-like componen s. Nucleoli consis o
se e al RNAs complex o di e en ibonucleop o eins (RNA binding
p o eins o RBPs) in ol ed in ibosome p oduc ion and many o he
unc ions. I has been p oposed ha he liquid-like beha io o ibonu-
cleop o eins enables he spon aneous o ma ion o MLOs since pu i ied
nucleola p o eins phase sepa a e in o d ople s esembling nucleoli
[27]. Ne e heless, he ep esen a ion o nucleoli as pu ely liquid-like
compa men s has been challenged. A s udy conduc ed by he B ang-
wynne eam used quan i a i e imaging and ma hema ical modeling o
in es iga e he p ope ies o nucleola ma e ial and hei implica ion in
he nucleola o m and unc ion. Thei indings sugges ha he nucle-
ola shape is uned by he cons ained mobili y o RNA and mechanical
cons ain s a ising om he su ounding he e och oma in [30]. The e-
o e, nucleoli can be conside ed complex luids wi h iscoelas ic p op-
e ies, exhibi ing ea u es o bo h solids and liquids [30].
The nucleolus con ains sepa a ed laye s o in e nal sub-
compa men s, owing o di e ences in sequence-encoded biophysical
p ope ies o i s mac omolecule cons i uen , e.g., d ople su ace ension
[27]. The mammalian nucleolus is in e nally o ganized in a leas ou
K. Gonz´
alez-A zola
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
3
concen ic sub-compa men s: he inne mos s uc u e is he ib illa
cen e (FC), ollowed by he dense ib illa componen (DFC), hen by
he g anula componen (GC), whe eas he ou e mos laye co esponds
o a ing o condensed pe inucleola ch oma in (PC) (Fig. 2). The FC and
DFC o m unc ional uni s o modules wi h mul iple copies imme sed in
a unique GC [31], and each FC-DFC uni con ains wo o h ee an-
sc ip ionally ac i e DNAs a he FC/DFC bo de [32] (Fig. 2).
In e es ingly, mos euka yo ic nucleoli, including hose in yeas ,
plan s, all in e eb a es, and some e eb a es, ha e only wo main
sublaye s: a cen al ib illa zone and a pe iphe al g anula egion
[33,34]. I is belie ed ha du ing e olu ion, he ib illa zone o nucleoli
sepa a ed in o wo domains, leading o he o ma ion o he FC and he
DFC [33]. Consequen ly, he di e en ia ed FC sublaye eme ged in
ep iles a he ansi ion be ween he anamnio es and amnio es,
ollowing a subs an ial inc ease in he size o he DNA in e genic egion
[33,34]. A sel -assembling sca old p o ein, T eache Collins-
F ancesche i synd ome (TCOF1, a.k.a. T eacle), is hough o be
esponsible o FC o ma ion and may p o ide insigh in o he eme -
gence o he FC ac oss e olu ion [35,36]. The mul ilaye ed nucleola
s uc u e has been conse ed in mode n Euka yo es. The eme gence o
addi ional nucleola sub-compa men s may ha e impa ed egula o y
unc ions o nucleoli, o e ing he po en ial o apid esponses o
en i onmen al s imuli h ough he selec i e seques a ion o esponse
ac o s [34].
Nucleola sub-compa men aliza ion pe ec ly e lec s he ole o his
o ganelle in he di e en s eps o RNA syn hesis and p ocessing, as well
as p e- ibosomal subuni assembly, occu ing du ing ibosome biogen-
esis (Fig. 3). FC holds he DNA ansc ip ion machine y, including RNA
polyme ase I (POL-I), UBF, and p omo e selec i i y ac o complex
(SL1), among o he ac o s [7,22]. DFC docks ac o s o he ea ly s ages
o p e- RNA p ocessing, such as small nucleola ibonucleop o eins
(snoRNPs), small nucleola RNAs (snoRNAs), ib illa in (FBL), and
o he s [7,22]. As nascen RNAs eme ge om he bo de be ween FC and
DFC (whe e POL-I is mos abundan ), hey bind o FBL [31]. FBL is a sel -
associa ed RBP esponsible o he ea ly p ocessing and me hyla ion o
RNAs [37], which is endowed wi h an N- e minal glycine- and a ginine-
ich (GAR) domain ha con ains se e al in insically diso de ed egions
(IDRs) and a C- e minal me hyl ans e ase egion which, in u n, com-
p ises an RNA binding domain (RBD) [32]. The binding o he newly
syn hesized p e- RNAs o he C- e minal RBD o FBL (along wi h he sel -
assembly endency o his la e ia i s GAR domains) p omo es FBL
phase sepa a ion, he es ablishmen o he DFC phase, and he p e-
RNAs so ing in o he DFC o ini ia e hei p ocessing [32]. Once he
polycis onic p e- RNA clea ages in o 28S, 18S, and 5.8S RNAs, hese
Fig. 1. Schema ic ep esen a ion o DNA genes in mammalian cells.
The DNA gene a ay is loca ed a he nucleola o ganize egions (NORs). I is posi ioned be ween elome es and cen ome es on he sho a ms (p-a ms) o he
ac ocen ic ch omosomes 13, 14, 15, 21, and 22 in humans. They a e lanked by he e och oma ic dis al and p oximal junc ions (DJ and PJ, espec i ely). NORs can
be obse ed as ach oma ic gaps on ch omosomes due o educed condensa ion o DNA. NORs a e si ua ed be ween elome ic and cen ome ic he e och oma in. Each
DNA epea uni (~43 kb) consis s o a ansc ibed egion (encoding he 45S p e-mRNA o 18S, 5.8S, and 28S ma u e RNAs) and an in e genic space (IGS). The
DNA ansc ibed egion in humans is 13 kb, while he IGS egion has 30 kb. The coding egion is lanked by ex e nal ansc ibed space s (5
′
ETS and 3
′
ETS), and DNA
genes a e sepa a ed by in e nal ansc ibed space s (ITS1 and ITS2). A ansc ip ion e mina ion si e is loca ed downs eam o he ansc ibed egion. The IGS
comp ises a p e- RNA (gene) p omo e and a space p omo e . Dis al o he space p omo e a e si ua ed enhance elemen s. The IGS egion may con ain
nucleosomes.
Fig. 2. In e nal o ganiza ion o mammalian nucleoli.
Quad ipa i e s uc u e o a ma u e nucleolus in in e phase: ib illa cen e (FC,
da k blue), dense ib illa componen (DFC, medium blue), g anula componen
(GC, ligh blue), and pe inucleola ch oma in (PC, g ay). The FC-DFC modules
a e p esen in se e al dozen copies in humans and a e imme sed in a single GC
sub-compa men . Each FC-DFC bipa i e s uc u e con ains wo o h ee an-
sc ip ionally ac i e DNAs (o ange) a he FC/DFC in e ace. Dis al junc ion
(DJ) sequences loca e a he PC nucleola laye . (Fo in e p e a ion o he e -
e ences o colou in his igu e legend, he eade is e e ed o he web e sion
o his a icle.)
K. Gonz´
alez-A zola
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
4
la e a e modi ied by snoRNPs and addi ional p ocessing ac o s [22].
Al hough he RBD o FBL is si ua ed inside i s me hyl ans e ase egion,
he me hyla ion unc ion o his la e domain is no ele an o p e-
RNA so ing [32]. Supe - esolu ion elec on mic oscopy analyses
ecen ly e ealed ha FBL and o he ibosome assembly ac o s o m a
ne wo k o 18–24 egula ly spaced clus e s o mini condensa es in o he
DFC [31,32]. Among hese ac o s a e non-his one ch omosome p o ein
2-like 1 (NHP2L1) and dyske in pseudou idine syn hase 1 (DKC1) [32].
The signi icance o such nanosized clus e s inside he DFC is unknown,
bu hei p esence sugges s compa men alizing he di e en RNA p o-
cessing eac ion s eps.
The ou e GC encloses he FC-DFC uni s, and i is he place whe e he
la e s ages o ibosome syn hesis ake place, i.e., he la e RNA p o-
cessing and he p e- ibosomal subuni assembly. The 5.8S and 28S
RNAs in he GC assemble wi h he 5S RNA and ibosomal p o eins
(RPs) o o m he 60S subuni . In con as , he 18S RNA and o he RPs
build he 40S ibosome subuni [22] (Fig. 3).
Nucleophosmin (NPM, a.k.a. NPM1 and B23) is an abundan his one
chape one esiding in he GC ha s imula es he DNA ansc ip ion by
egula ing he his one densi y a ound he DNA genes [38]. Besides i s
ole in he nuclea expo o he 60S and 40S ibosomal subuni s [39],
NPM has la ely been assigned a ole in sepa a ing nascen 60S pa icles
om he ch oma in ac ion o he FC-DFC module owa ds GC, whe e
u he ma u a ion p oceeds [40]. This chape one encompasses a
s uc u ed N- e minal oligome iza ion domain, a middle-diso de ed e-
gion esponsible o his one chape oning, and a olded C- e minal
nucleic acid binding domain [41].
In addi ion o ibosome syn hesis, mRNA anspo , ch oma in
emodeling, and DNA epai [41], NPM egula es he al e na i e eading
ame (ARF)-p53 umo supp ession pa hway [42,43]. Pionee ing bio-
physical app oaches pe o med by K iwacki’s g oup demons a ed ha
NPM d ama ically con ibu es o he nucleola sub-s uc u e using i s
abili y o LLPS [44,45]. To d i e phase sepa a ion, NPM displays a
succession o h ee acidic and wo basic ac s, mos loca ed in he
middle IDR and one in he pen ame ic domain, p o iding a solid in e -
ac ion mul i alency. Thus, mul i alen in e ac ions o NPM wi h
nucleola p o eins con aining a ginine- ich (R- ich) linea mo i s and
RNA con e he liquid-like ea u e o he GC [44]. A ecen s udy ound
ha in addi ion o R-mo i s, lysine- ich (K- ich) p o eins also igge
NPM phase ansi ions o egula e he a icking o nucleola p o eins
[43].
Ano he abundan p o ein in he GC is nucleolin (NCL), a his one
chape one ha acili a es DNA ansc ip ion by p omo ing a euch o-
ma ic s a e [46]. NCL consis s o h ee acidic s e ches a he amino-
e minal end, ou RBDs, and a ca boxy- e minal GAR domain [47].
Acco ding o a ecen in es iga ion, he GAR domain o NCL d i es i s
subcellula localiza ion in he axon o neu ons and he plasma mem-
b ane o umo cells, hus con ibu ing o placing NCL wi hin he
nucleolus [48]. Conside ing he in ol emen o he GAR domains o FBL
in he phase ansi ions a he nucleola DFC, i is emp ing o hypo h-
esize a simila ole o he GAR domain o NCL in he GC, in addi ion o
he phase ansi ions accomplished by NPM.
The na u e and exac cellula unc ion o he ou h nucleola laye
(PC) is s ill con o e sial. 3D luo escence in si u hyb idiza ion (3D
FISH) analysis has led o he p oposal ha PC concen a es inac i e
ch omosomal egions en iched in genes in ol ed in esponse o o he
o ganisms (de ensin, immunoglobulin, and T-cell ecep o genes), sen-
so y pe cep ion (ol ac o y genes), issue de elopmen and emb yo im-
plan a ion, as well as a la ge numbe o snoRNA genes, 5S DNA and
RNA genes [49]. Such gene amilies posi ioned a PC a e a anged in
la ge gene clus e s and may be issue-speci ic exp essed [49]. Acco ding
o he appa en inac i e ch oma in s uc u e o PC, se e al ep essi e
Fig. 3. Nucleola o ganiza ion and i s unc ion in ibosome biogenesis.
The o e all s uc u e o he mammalian nucleolus shows he in e nal ib illa cen e (FC), a dense ib illa componen (DFC), and an ex e nal g anula componen
(GC), all o hem su ounded by condensed pe inucleola ch oma in (PC). RNA polyme ase-I (POL-I) ansc ibes he DNA genes o o m a 47S p e- RNA a he FC-
DFC bo de . A p e-ini ia ion complex o med by POL-I, ansc ip ion ac o ups eam binding ac o (UBF), he p omo e selec i i y ac o complex (SL1), an-
sc ip ion ini ia ion ac o -IA (TIF-IA), and T eacle is equi ed o DNA clus e ansc ip ion. Type-I/II opoisome ases (TOPO I/II) ac ion is also needed o ini ia e
DNA ansc ip ion. In he DFC, p e- RNA is u he p ocessed in ma u e 18S, 5.8S, and 28S RNAs. DFC ha bo s small nucleola RNAs (snoRNAs), small nucleola
ibonucleop o eins (snoRNPs), ib illa in (FBL), and o he ac o s in ol ed in p e- RNA p ocessing. In he nucleoplasm, he 5S DNA and he ibosomal p o eins (RPs)
genes a e ansc ibed by RNA polyme ase III (POL-III) and RNA polyme ase II (POL-II), espec i ely. In he GC, RPs associa e wi h RNAs o o m he ibosomal
subuni s. IGS: in e genic space .
K. Gonz´
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BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
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his one ma ks can be de ec ed (H3K27me3, H3K9me3, and his one H4
Lys20 ime hyla ion o H4K20me3), whe eas ac i e his one ma ks,
such as H3K4me3, a e nea ly deple ed a hese egions [49]. Al hough
he ch oma in adjacen o nucleoli, he so-called nucleola -associa ed
domains o NADs, displays he e och oma ic ea u es, o he nucleola
genome o ganiza ion landscapes canno be uled ou due o me hodo-
logical limi a ions o NAD iden i ica ion [50]. Cu iously, one egion
loca ed o he pe iphe y o he PC is lanking he NORs, he DJ [23]
(Fig. 2). These sequences posi ioned in he PC display ansc ip ionally
ac i e ch oma in ea u es, hus sugges ing a ole o DJs in nucleola
o ma ion and unc ioning [51–53]. DJ egions also o ganize he DNA
a ay when i is damaged, implying hei pa icipa ion in DNA gene
s abili y, as men ioned below.
Spa ial mapping o he nucleola p o eome e ealed ha some
nucleola p o eins ancho he nucleolus o he PC and o m a “nucleola
im” [54]. The nucleola im holds se e al p o eins, such as ma ke o
p oli e a ion Ki-67 (MKI67), which is needed o p ope ch omosome
seg ega ion du ing mi osis, and guanine nucleo ide binding p o ein-like
3 (GNL3) ha is equi ed o cell p oli e a ion, among o he ac o s [54].
The quad ipa i e s uc u e o he nucleolus is isible by elec on mi-
c oscopy (EM), bu he o he la ely desc ibed nucleola sub-s uc u es
a e no easily obse ed by EM; hese include he pe iphe y o DFC
(in ol ed in he ma u a ion o RNAs [36,55]) and he nucleola im
whose biochemical unc ion emains a mys e y.
Un il ecen ly, i was belie ed ha NPM and NCL in he GC and FBL
in he DFC we e he only nucleola p o eins s ongly implica ed in
nucleola assembly; howe e , o he ac o s may con ibu e o he
nucleola a chi ec u e. An in eg a i e so wa e app oach calcula es he
iNo sco e, de e mining he deg ee o nucleola dis up ion a e deple ing
speci ic nucleola p o eins [56]. This analysis de e mined ha e y ew
RPs a e equi ed o main ain he nucleola s uc u e, and only la e-
assembled RPs o he 60S la ge ibosomal subuni signi ican ly
con ibu ed o nucleola s uc u e [56]. Among 60S RPs, ibosomal
p o ein la ge 5 (RPL5, a.k.a. uL18) and RPL11 (a.k.a. uL5) a e essen ial
o nucleola s uc u e main enance [56–58]. Cu iously, RPL5 and RPL11
a e necessa y o ac i a ing he p53 pa hway upon dis up ion o ibo-
some biogenesis [59]. RPL5 and o he R- ich RPs, such as guanine
nucleo ide binding p o ein-like 2 (GNL2), su ei 6 (SURF6) and RPL23
(a.k.a. uL14), display liquid phase ansi ions wi h NPM [44]. Rema k-
ably, a new in es iga ion has shown ha he e och oma ic p o eins,
pa icula ly he e och oma in p o ein 1
α
and β (HP1
α
and HP1β), ensu e
he s uc u al in eg i y o nucleoli [60]. This ag ees ha HP1
α
igge s
LLPS-d i en he e och oma in o ma ion [61,62]. A no el supe -
esolu ion mic oscopy (SRM) analysis e ealed ha nucleola p o ein
10 (NOL10) cons i u es he “skele al s uc u e” o he nucleola GC and
ha his p o ein is indispensable o main aining he s uc u e o he
nucleolus [63]. Recen ad ancemen s in echniques such as SRM and
co ela i e ligh and elec on mic oscopy (CLEM) ha e enabled he
imaging o indi idual p o eins wi hin nucleola sub-compa men s [63].
Fu he mo e, he use o high-speed a omic o ce mic oscopy (HS-AFM)
has allowed he dynamic imaging o he globula egions and he un-
s uc u ed ails o nucleola in insically diso de ed p o eins (IDPs)
[63]. In he u u e, he applica ion o high- esolu ion echniques is ex-
pec ed o u he imp o e ou unde s anding o he sub-s uc u e o he
nucleolus.
Besides he pa icipa ion o phase-sepa a ed p o eins in nucleola
s uc u e, i has been ecen ly poin ed ou ha ce ain RNAs con ibu e
o his p ocess, sugges ing ha nucleola a chi ec u e is coo dina ed
wi h RNA p ocessing and ma u a ion [64]. A speci ic RNA in e me-
dia e, 27SA
2
, is c ucial in main aining he sphe ical shape o nucleoli in
C. elegans [64].
2.2. Ribosome syn hesis: he canonical nucleola ole
Al hough ibosome syn hesis is p ese ed ac oss all domains o li e,
he p esence o a nucleolus has been only documen ed in Euka yo es
and, mos ecen ly, in A chaea [65]. In he nucleolus, POL-I ansc ibes
he DNA genes encoding he small subuni S- RNA (18S) and he la ge
subuni 5.8S and 28S L- RNAs as a single polycis onic p ecu so (47S
p e- RNA).
Each DNA epea uni a NORs consis s o a p e- RNA coding egion
and an in e genic space (IGS). The p e- RNA coding egion gene a es
he 47S p e- RNA, which is u he p ocessed in o h ee ma u e RNA
molecules: 18S, 5.8S, and 28S (Fig. 1). The DNA coding sequences a e
highly conse ed ac oss species. S ill, he IGS egion is less conse ed
and con ains egula o y elemen s, such as wo p omo e s and se e al
epe i i e enhance elemen s [66] (Fig. 1). A he IGS, he gene p omo e
con ols he polycis onic 47S p e- RNA ansc ip ion. In con as , he
space p omo e encodes non-coding RNAs likely in ol ed in gene
silencing [66]. No ably, long non-coding RNAs (lncRNAs) a e p oduced
om he IGSs o DNA genes, and hey seem o ha e egula o y oles in
esponding o en i onmen al s esso s, as desc ibed below. Thus, hese
nucleola lncRNAs allow cellula adap a ion o s ess by educing he
in ense ansc ip ion o DNA genes and causing p o ein seques a ion
wi hin he nucleolus [67].
POL-I ansc ip ion equi es he o ma ion o a p eini ia ion complex
on he p e- RNA p omo e , including binding he nucleola ansc ip ion
ac o UBF and SL1, among o he s [13]. The ansc ip ion ini ia ion
ac o IA (TIF-IA; a.k.a. RRN3) in e ac s wi h POL-I, enabling he POL-I
binding o SL1 [68]. SL1 complex con ains he TATA-binding p o ein
(TBP) and i e TBP-associa ed ac o s: TAF1A, TAF1B, TAF1C, TAF1D
and TAF12 [69]. Ano he i al ansc ip ion ac o is UBF, which dis-
places his one H1 and main ains an open ch oma in con igu a ion sus-
cep ible o binding o addi ional ac o s [70]. Se e al o he ac o s a e
associa ed wi h he p eini ia ion complex o ini ia e ansc ip ion, as is
he case o TOPO I and II, which emo e DNA supe coils a he DNA
p omo e [71,72], and T eacle ha enables he ec ui men o POL-I and
UBF o he p e-ini ia ion complex [73]. T ansc ip ion e mina ion ac o
1 (TTF1) con ols ansc ip ion e mina ion and s ops elonga ion by
POL-I. Genes encoding he 5S RNA a e also p esen in andem epea s,
bu in a sepa a e genomic loca ion, on he dis al end o ch omosome 1
[66]. These genes a e ansc ibed by RNA polyme ase-III (POL-III)
ou side o he nucleolus [66]. Inco po a ion o he 5S RNA in o he p e-
ibosome is poo ly unde s ood. A ecen s udy has p oposed ha nascen
5S RNA binds o RPL5, RPL11, and o he p o eins wi hin he nucleo-
plasm [74]. The 5S ibonucleop o ein complex is hen ec ui ed by
nucleola ac o s ( ibosome p oduc ion ac o 2 o RPF2, and ibosome
biogenesis egula o y p o ein homolog o RRS1) o o m a hexame ic
complex ha is ul ima ely inco po a ed in o he p e-60S subuni [74].
In addi ion o emo ing he ansc ibed space egions h ough en-
donucleoly ic and exonucleoly ic clea ages, RNAs a e submi ed o
u he modi ica ions in he nucleolus (me hyla ion and pseudou -
idyla ion) by snoRNPs [75]. The nascen p e- RNAs associa e co-
ansc ip ionally wi h some ibosomal p o eins and snoRNPs o o m
la ge pa icles in which p e- RNAs a e olded and modi ied o yield he
ma u e RNAs [76]. In he nucleoplasm, RNA polyme ase-II (POL-II)
ansc ibes he genes encoding ibosomal p o eins (RPs) and assembly
ac o s, which a e ansla ed in o he cy oplasm and eimpo ed o he
nucleus o assembly. Mos RPs a e anspo ed o he nucleus by mul-
iple ka yophe ins o impo ins, which ecognize he nuclea localiza-
ion signals o hei ca go p o eins and ca y hem h ough he nuclea
po e complex [77]. Addi ionally, NCL and NPM assis in a ge ing RPs o
he nucleolus [78,79]. POL-II also ansc ibes abou 200 snoRNAs in he
nucleoplasm, pa icipa ing in p e-RNA p ocessing [80]. SnoRNAs asso-
cia e wi h nucleola p o eins 56 and 58 (NOP56 and NOP58), FBL,
among o he s, which p omp s hei nucleola a ge ing [81]. In e es -
ingly, ch omosome egion main enance 1 (CRM1) p omo es he nucle-
ola a ge ing o snoRNAs ia he exposi ion o he nucleola localiza ion
sequence (NoLS) o NOP58 [81].
La e , he 80 RPs ha o m he 60S la ge and 40S small ibosomal
subuni s, 47 and 33 RPs, espec i ely [49], en e he nucleolus and
associa e wi h he ma u e RNAs o gene a e he p ecu so s o he
K. Gonz´
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BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
6
ibosomal subuni s [82]. These a e hen expo ed o he cy oplasm
h ough he nuclea po e complex, o ming a ma u e ibosome o ini ia e
p o ein syn hesis (Fig. 3). In iguingly, a la es epo s a ed ha a
nucleola pool o ac i e POL-II binds o he IGS egions o DNA genes o
d i e hei exp ession [83].
Nucleoli a e wholly disassembled by he end o p ophase o p epa e
cell di ision, seg ega ing he daugh e cells’ cellula componen s.
Nucleola disassembly ini ia es when DNA ansc ip ion is abolished
h ough cyclin-dependen kinase 1 (CDK1)-media ed POL-I inac i a ion
[84]. The nucleolus o ma ion occu s a he end o mi osis (in elophase)
when p e-nucleola bodies con aining FBL, NPM, and ibosomal RNA
p ocessing p o ein 1 homolog A (RRP1A a.k.a. NOP52) a e ec ui ed o
NORs [85]. The la e a ises when DNA ansc ip ion esumes due o
dec eased CDK1 ac i i y, leading o u he e-ac i a ion o POL-I [65].
Du ing in e phase, mul iple nucleoli use o o m la ge nucleoli ha
con ain se e al NORs and a e su ounded by he e och oma in [52].
3. Nucleola s ess
3.1. The nucleolus as a cen al hub o cellula s ess esponse
coo dina ion
In ecen decades, a ious in es iga ions ha e demons a ed ha he
nucleolus is an o ganelle wi h mul iple unc ions. Al hough ibosome
biogenesis is he adi ional p ima y unc ion o he nucleolus, only ~30
% o nucleola p o eins a e associa ed wi h ibogenesis [7]. Va ious
p o eomic s udies ha e iden i ied nucleola ac o s in ol ed in many
o he mammalian p ocesses, i.e., cell cycle p og ession, p oli e a ion,
apop osis, and he esponse o di e en insul s [86–88]. The a ied
unc ions o o e 4500 nucleolus-associa ed p o eins, acco ding o he
la es upda e o he Nucleola P o eome Da abase (NOPdb3.0), a e
consis en wi h non-canonical addi ional oles o he nucleolus [86].
The nucleolus senses se e al ypes o cellula s ess, which u ns his
o ganelle in o a cen al hub o coo dina ing he subsequen esponse
[7].
Due o he absence o a delimi ing memb ane, he nucleoli compo-
si ion is highly a iable. Ag eeing o such a dynamic na u e, he
nucleolus is an ideal loca ion o apid cellula homeos asis egula ion
h ough he ansien seques a ion o c i ical egula o s o he cellula
s ess esponse, which o en esul s in nucleola changes in size, numbe ,
and p o ein composi ion on demand [11]. Impo an ly, gi en ha
ibosome biogenesis is di ec ly coupled wi h cell p oli e a ion, i is no
su p ising ha nucleoli play a c ucial ole in o ches a ing p ima y
cellula esponses o s ess. Thus, in acco dance wi h hei luid na u e,
nucleola componen s exhibi con inuous lux wi hin he nucleolus and
a e exchanged wi h he su ounding nucleoplasm unde a ious s ess
si ua ions.
The link be ween cellula s ess and nucleoli comes om he no ion
ha hese o ganelles di ec ly egula e he s ess- esponsi e ac o pa
excellence, p53 [89,90]. Expec edly, a common ea u e in many p53-
media ed cellula esponses is nucleola s uc u e diso ganiza ion
[10,90]. p53 is a mas e ansc ip ion ac o p edominan ly egula ed by
p o easomal deg ada ion media ed by i s ubiqui in ligase mu ine double
minu e 2 (MDM2, HDM2 in humans) [91]. Se e al kinds o cellula in-
ju ies induce edis ibu ion o he nucleoplasm o nucleola p o eins o
s abilize p53, which u he induces cell cycle a es o apop osis by
con olling dozens o a ge genes [92,93]. These nucleola p o eins
include NPM [93], RPL5 [94], RPL6 [95], RPL11 [96], RPL23 [97], i-
bosomal p o ein small 3 (RPS3) [98], RPS14 [99], RPS27-like [100] and
glioma umo -supp esso candida e egion gene 2 (GLTSCR2) [101].
The abo e-men ioned nucleola p o eins a e eleased om he nucleoli
o inhibi MDM2, hus leading o p53 s abiliza ion [93–101]. All his
ein o ces he idea ha nucleola p o eins ac i a e p53 and ha he
nucleolus is a senso o cellula s ess. Despi e his, g owing e idence
sugges s ha p53 is no he only s ess- esponsi e ac o ac i a ed by he
nucleolus [102]. This is pa icula ly impo an because many cance
cells con ain mu an p53 [103]. Such p53-independen mechanisms also
in ol e ibosomal p o eins and o he nucleola ac o s such as NPM and
ARF [102]. When eleased om nucleoli, RPL5, RPL11, and RPS14
inhibi he ac i i y o he ansc ip ional ac o c-Myc, hus p e en ing
he c-Myc-media ed ac i a ion o genes in ol ed in cell g own and
p oli e a ion [104,105]. Mo eo e , NPM and ARF ansloca e om he
nucleolus and bypass p53 o di ec ly egula e apop osis. Following DNA
inju ies, NPM eloca es o he cy oplasm o seques e BCL2-associa ed x
p o ein (BAX), hus a oiding he p o-apop o ic e ec o BAX [106]. In
esponse o geno oxic s ess, ARF ac i a es a axia elangiec asia mu a ed
(ATM)/a axia elangiec asia and Rad3- ela ed p o ein (ATR) signaling
pa hways, hus inducing p53-independen cell cycle a es [107]. On he
o he hand, o he ansc ip ion ac o s, such as he plan -speci ic amily
called NAM/ATAF/CUC (NAC), espond o nucleola s ess caused by
pe u ba ions o ibosome biogenesis in plan s, which lack p53 [108].
Rema kably, some biomolecula condensa es a e o med wi hin he
nucleola space and play a c ucial ole in p o ein seques a ion du ing
s ess ul condi ions [109]. Some a e agg ega es simila o amyloid pla-
ques, a.k.a. amyloid bodies o A-bodies, o igina ing om lncRNAs
ansc ibed om he ibosomal IGSs [110]. A-bodies a e induced in
esponse o a ious en i onmen al s imuli such as hea shock, acidosis,
and o he s ess ul si ua ions [109,111]. Du ing s ess, clus e s o
lncRNAs wi h dinucleo ide epea s (CU o AG) accumula e in he
nucleola a ea. The low-complexi y sequences o lncRNAs acili a e
cha ge-based in e ac ions wi h ca ionic p o eins o p oduce nucleola
liquid-like oci which seques e hese p o eins away om hei down-
s eam e ec o s [110]. Hea shock igge s he o ma ion o lncRNAs
loca ed 16 kb (IGS16RNA) and 22 kb (IGS22RNA) downs eam o he
DNA ansc ip ion s a si e (TSS) [112]. In con as , acidosis causes he
accumula ion o a ansc ip loca ed 28 kb (IGS28RNA) downs eam o
he TSS [112]. When he p o easome ac i i y in mammalian cells is
hampe ed, i causes he accumula ion o polyubiqui yla ed p o eins.
This leads o he o ma ion o RNA-p o ein agg ega es in he nucleola
space ha a e known as nucleola agg esomes [113,114]. These sub-
nucleola s uc u es con ain polyadenyla ed RNA, ubiqui in, and
se e al nucleoplasmic p o easome a ge p o eins. The la e includes
key ac o s in oncogenesis, cell cycle- egula ing cyclins and cyclin-
dependen kinases (CDKs), and s ess esponse kinases [114]. The e is
ano he class o sub-nucleola s uc u es called in anucleola bodies
(INBs). INBs a e ound in abundance in uns essed nucleoli and a e
loca ed nea DNA wi h he e och oma ic ea u es [115]. They appea o
egula e he ansc ip ion and main enance o DNA genes. INB o ma-
ion is enhanced by speci ic DNA-damaging condi ions like in a ed
i adia ion and e oposide o camp o hecin ea men [115]. Howe e ,
hese nucleola agg ega es do no dis up he s uc u al in eg i y o he
nucleolus. INBs con ain p o eins ha a e in ol ed in DNA epai and
eplica ion, p o ein u no e , RNA p ocessing, ch oma in o ganiza ion,
as well small ubiqui in-like modi ie 1 (SUMO 1) and SUMO 2/3 [115].
The close ela ionship be ween he nucleolus and s ess was
desc ibed in he middle o he las cen u y when nucleoli we e selec-
i ely i adia ed wi h ul a iole ligh , and cell cycle a es immedia ely
occu ed [116]. Since, a ha ime, he only known unc ion o he
nucleolus was he syn hesis o ibosomes, his led o he hypo hesis ha
de iciency in ibosomes o he ibosomal unc ioning is he cause o cell
cycle a es . Consequen ly, he e m “nucleola s ess” was adi ionally
used o e e o “ ibo oxic s ess” o “ ibosome biogenesis s ess.”
Nucleola s ess has jus eme ged as a no el concep , and i is now
e e ed o as di e se cellula insul s ha induce abno mali ies in
nucleola s uc u e and unc ion [6,10].
As poin ed ou be o e, many s esso s display a nucleola esponse
leading o he ini ia ion o he p53-dependen cell cycle a es o
apop osis, depending on he se e i y o he inju ies. Ne e heless, a
epo sugges s ha p53-de icien U2OS os eosa coma and HCT-116
colon cance cells a e also sensi i e o he nucleola s ess induced by
DNA inhibi ion [117]. In such cases, cell cycle a es elies on he
deg ada ion o E2F ansc ip ion ac o 1 (E2F1) [117]. In addi ion, p53-
K. Gonz´
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BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
7
knockou e y h oid cells unde going de ec s in ibosome syn hesis we e
able o block cell cycle p og ession due o he s abiliza ion o he cell
cycle inhibi o p27 [118]. In con as , cells lacking p53 p o ein
exp ession submi ed o dis up ion o ibosome biogenesis displayed
G1/S a es o apop osis due o p53-independen ac i a ion o p21
exp ession [119]. In addi ion o ac i a ing p21, nucleola s ess igge s
addi ional mechanisms ha lead o cell cycle a es . One such mecha-
nism in ol es he ansloca ion o ibosome p o ein la ge 3 (RPL3, a.k.a.
uL3) in o he nucleoplasm ollowing a geno oxic ea men [120]. The e,
RPL3 binds o poly ADP- ibose polyme ase 1 (PARP-1), hus p e en ing
he binding o he la e o he E2F1 p omo e and a oiding he an-
sc ip ion ac o ac i a ion. As E2F1 is esponsible o ac i a ing he
ansc ip ion o genes equi ed o en y in o he la e G1/S phase, his
leads o cell cycle a es [120]. Mo eo e , RPL3 nega i ely egula es
cyclin D1 by educing i s mRNA and p o ein le els, hus inhibi ing he
p og ession h ough he G1 phase [120]. S ikingly, RPL3 exp ession is
down egula ed in colon cance cells [121], which makes some chemo-
he apeu ic agen s ine ec i e [122]. Mo eo e , RPL3 ac s as an inhibi-
o o he au ophagic p ocess in colon umo issues [122]. Au ophagy
has been linked o bo h umo igenesis and chemo he apeu ic esis ance
[123]. The e o e, RPL3 imp o es he ac i i y o many an icance d ugs
hanks o i s p o-apop o ic and an i-au ophagic ac i i ies in p53 de i-
cien cance cells [122]. These epo s sugges ha p53-independen
mechanisms igge cell cycle p og ession blocking o cell dea h unde
nucleola al e a ions. The p53-dependen and -independen esponses o
nucleola al e a ions a e ex ensi ely e iewed in [5,124]. Hence, a mo e
p ecise desc ip ion o nucleola s ess can be anno a ed as s ess ul
cellula e en s ha impac nucleola mo phology and unc ions, ul i-
ma ely ac i a ing p53 o o he s ess signaling pa hways o induce cell
cycle a es o cell dea h.
Acco ding o Yang e al., he ac o s inducing nucleola s ess can be
classi ied as canonical and non-canonical [10]. The o me s impai o
al e any s ep o he ibosome biogenesis; hus, canonical nucleola
s ess induce s a e o en POL-I inhibi o s [125] o ac o s inducing
abe an exp ession o nucleola p o eins [126]. On he o he hand, he
non-canonical signals include a ious gene al cellula insul s ha a ec
non- ibosomal unc ions, e.g., UV ligh , hea shock, hypoxia, ee adi-
cals, nu ien dep i a ion, and many o he s [10]. In any case, nucleola
s ess is o en e lec ed in d as ic changes in he s uc u e and compo-
si ion o he nucleolus (Table 1).
3.2. Canonical nucleola s ess
Cellula s esso s ha al e any o he mul iple s eps o ibosome
biogenesis may lead o canonical nucleola s ess, e iewed below
(Fig. 4).
3.2.1. DNA ansc ip ion inhibi ion
A well-desc ibed example o canonical nucleola s ess is he nucle-
ola seg ega ion induced by ac inomycin D. This d ug in e cala es wi h
bo h double and single-s anded DNA, mainly a GC- ich egions which
a e p esen a a high equency in DNA genes, hus pausing POL-I and
blocking DNA ansc ip ion elonga ion [131]. No ewo hy is ha
ac inomycin D selec i ely inhibi s POL-I and induces nucleola s ess a
a low concen a ion (5 nM). In con as , high doses (>30 nM) may also
p oduce DNA damage and nuclea ansc ip ion inhibi ion [161].
O he in e cala ing agen s a ec ing nucleola s uc u e a e doxo u-
bicin and mi oxan one. These compounds es ablish co alen complexes
be ween DNA and ype II opoisome ases, inhibi ing he la e and
p e en ing he DNA ansc ip ion ini ia ion by POL-I [129,130].
Mi omycin C is ano he in e cala ing d ug ha inhibi s DNA an-
sc ip ion by an unknown mechanism [131]. Me ho exa e is a ola e
analog ha inhibi s he syn hesis o pu ine and py imidine, he eby
impeding he ansc ip ion ca ied ou by POL-I [131,132].
The pla inum compounds oxalipla in and cispla in a e ex ensi ely
used cance chemo he apeu ic agen s, and hey ha e been p oposed o
Table 1
E ec s o canonical and non-canonical signals igge ing nucleola s ess.
S ess ype Cause E ec on nucleola
s uc u e
Re .
Inhibi ion o
DNA
ansc ip ion
Ac inomycin D (low
dose)
Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[127,128]
Doxo ubicin Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[129,130]
Mi oxan one Nucleola caps [129,130]
Mi omycin C Nucleola caps [131]
Me ho exa e Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[131,132]
Oxalipla in Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132]
Cispla in
(high dose)
Nucleola caps,
ansloca ion o
nucleola p o eins o he
nucleoplasm
[132,133]
BMH-21 Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[134,135]
Me a es in Nucleola dis up ion [136]
CX-3543 (qua loxin) No de e mined [137]
CX-5461
(pidna ulex)
No de e mined [138]
Ellip icines No de e mined [139]
PMR-116 No de e mined [131,140]
Sodium ce i as a in No de e mined [141]
Aminoac idines Nucleola dis up ion [142]
Gold nanopa icles Redis ibu ion o
nucleola p o eins
[143,144]
Inhibi ion o
ea ly RNA
p ocessing
Camp o hecin and
de i a i es
In anucleola bodies [115]
Nucleola caps,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132]
Fla opi idol Nucleola necklace,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132]
Rosco i ine Nucleola necklace,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132]
DRB Nucleola necklace,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132,145]
TBA Redis ibu ion o
nucleola p o eins
[146]
Inhibi ion o
la e RNA
p ocessing
E oposide In anucleola bodies [115]
Nucleola spo s,
ansloca ion o
nucleola p o eins o
nucleoplasm
[132]
MG-132 Nucleola agg esomes [114]
None [132,147]
Homoha ing onine None [132]
Cycloheximide None [132]
5- luo ou acil None [132]
Inhibi ion o
subuni
assembly o
ma u a ion
Diazabo ine None [148]
Ribozinoindole-1 None [149]
Nuclea expo
inhibi ion
Lep omycin B Changes in he nucleola
composi ion
[150]
(con inued on nex page)
K. Gonz´
alez-A zola
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
8
igge nucleola s ess by DNA ansc ip ion inhibi ion [132]. How-
e e , ecen s udies ha e highligh ed ha , a clinically ele an doses,
oxalipla in causes cell dea h ia ibosome biogenesis s ess, whe eas
cispla in induces cy o oxici y ia he DNA damage esponse [162–164].
A high doses (100
μ
M), cispla in induces he o ma ion o adduc s wi h
DNA and UBF, hus hijacking UBF and ep essing DNA ansc ip ion
[11,133]. Howe e , a a low concen a ion (6.25
μ
M), cispla in nei he
inhibi s DNA ansc ip ion no induces nucleola s ess [164]. This
di e en d ug mechanism o cell dea h igge ing may elucida e he
dis inc clinical implemen a ion o pla inum analogs: cispla in is mainly
employed in b eas and lung umo s, while oxalipla in is used almos
solely in colo ec al and o he gas oin es inal cance s [162]. The
mechanism by which oxalipla in induces ibosome biogenesis s ess is
no ully unde s ood o da e, bu i is hough ha i also in ol es UBF
seques a ion [11].
A high- h oughpu sc eening o compounds wi h an i umo ac i i y
led o he iden i ica ion o he d ug BMH-21 as a po en ac i a o o p53
wi hou igge ing he cellula DNA damage esponse [134]. This
in e cala ing compound binds GC- ich sequences o DNA, hus hin-
de ing POL-I ansc ip ion. In addi ion, BMH-21 [135], aminoac idines
[165], he nandonine [166], and sempe i ine [167] p omo e he
p o easome-media ed deg ada ion o one o he POL-I ca aly ic subuni s
(RNA polyme ase I subuni A o RPA194). Me a es in is ano he an i-
cance d ug ha e ec i ely supp esses me as asis by hinde ing DNA
ansc ip ion and dis up ing he nucleola s uc u e [136].
The an icance d ug CX-3543 dis up s complexes be ween NCL and
DNA G-quad uplex, leading o he inhibi ion o elonga ion by POL-I
[137]. A molecule called CX-5461, ano he luo oquinolone de i a i e
wi h an i umo al po en ial, in e e es wi h he in e ac ion be ween he
SL1 complex and POL-I, hus blocking he p e-ini ia ion complex o -
ma ion [138]. Signi ican ly, CX-5461 has been ecen ly app o ed by he
Ame ican Food and D ug Adminis a ion (FDA) o he as - ack
p og am o ea b eas cance [11]. Howe e , i s an i umo al e ec is
hough o be ela ed o i s abili y o inhibi opoisome ase II a he han
a POL-I blocking e ec [168]. Ellip icines a e plan alkaloids used as
an i umo al agen s ha ha e been ound o inhibi POL-I-media ed
ansc ip ion since hey cause SL1 dissocia ion om he DNA p o-
mo e [139]. The newly de eloped d ug PMR-116 by Pime a The a-
peu ics has high umo ac i i y and g ea po en ial as a POL-I inhibi o ;
howe e , i s inhibi o y mechanism emains unclea [131,140]. Also
ecen ly, an al eady FDA-app o ed compound, sodium ce i as a in, has
been es ed as a POL-I inhibi o due o i s abili y o in e e e wi h he
polyme ase’s in e ac ion wi h TIF-IA, hus gi ing ise o DNA an-
sc ip ion blockage [141]. In addi ion, apamycin, a well-known inhibi-
o o he mammalian a ge o he apamycin (mTOR) pa hway,
impedes phospho yla ion o TIF-IA, hus in e e ing wi h he POL-I
ansc ip ion machine y [169].
POL-III ansc ibes he 5S RNA, making i a easible selec i e a ge
o ibosome syn hesis inhibi ion [170]. New in es iga ions showed ha
he na u al lac one ip olide dis up s he o ma ion o he ansc ip ion
ac o -IIIB (TFIIIB) complex ( he main POL-III ansc ip ion ac o ) a 5S
DNA p omo e s, hus inhibi ing 5S RNA ansc ip ion [170].
One common e ec o DNA ansc ip ion inhibi ion is a nucleola
diso ganiza ion cha ac e ized by he condensa ion and sepa a ion o he
nucleolus in o bipa i e s uc u es composed by he FC and GC, o ming
s uc u es a ound he nucleola emnan , he so-called nucleola caps
[127] (Fig. 5). Nucleola caps a e dynamic s uc u es in which he FC
and DFC a e in e ed and su ounded by he GC. They a e hallma ks o
he ansc ip ional shu down and o en con ain nucleola componen s,
such as DNA, FBL, UBF, and nucleoplasmic RBPs [127].
Gold nanopa icles ha e been ound o in e e e wi h he ansc ip-
ion o DNA and comp omise RNA p ocessing [144]. These nano-
pa icles a e ine and biocompa ible, making hem p omising ools o
cance he apy. Due o hei abili y o induce pho o he mal killing
p e e en ially in umo al cells, gold nanopa icles could educe he se-
e e side e ec s o o he an icance ea men s [171]. Indeed, a s udy
has shown ha inco po a ing gold nanosphe es combined wi h mild
hea s ess can educe he iabili y and p oli e a ion o b eas cance
cells [143].
3.2.2. Al e a ions in RNA p ocessing, ibosomal assembly, o
ibonucleop o ein anspo
P ocessing he 47S RNA polycis onic p ecu so equi es se e al
s eps o ob ain he ma u e RNAs. Camp o hecin, opo ecan, and i ino-
ecan a e ype-I opoisome ase inhibi o s ha mainly dis u b ea ly
s ages o he RNA p ocessing, measu ed as a de ec i e gene a ion o he
32S RNA in e media e, while ba ely a ec ing DNA ansc ip ion
[132]. Ac idine de i a i es also abolish he ea ly p ocessing o RNAs
[142].
O he compounds also a ec ing ea ly RNA p ocessing a e he CDK
inhibi o s la opi idol and osco i ine [132]. The exac mechanism o
hese inhibi o s is no ye ully unde s ood. I was hough ha i elied
on down egula ion o he U8 snoRNA, essen ial o 3
′
RNA p ocessing,
upon CDK9 inhibi ion [172]. Howe e , new esea ch unde lined he
in ol emen o nucleola POL-II and CDK9 in imp o ing he POL-I-
media ed ansc ip ion by coun e ac ing he ad e se e ec s o sense
in e genic non-coding RNAs (sincRNAs) p oduced by POL-I [83], which
expands he ole o CDK inhibi o s beyond he p ocessing o RNA. The
nucleo ide analog 5,6-dichlo o-1-β-D- ibo u anosylbenzimidazole
(DRB) is a POL-II inhibi o whose ac i i y depends on casein kinase II
(CKII) [173]. DRB has been ound o in e e e wi h he ea ly p ocessing
o RNA, hus p oducing nucleola s ess [132,145]. When ea ly RNA
p ocessing is impai ed, bu he DNA ansc ip ion emains ac i e, a
di e en nucleola e-o ganiza ion appea s, he so-called nucleola
necklace [145] (Fig. 5). Each necklace bead co esponds o he FC-DFC
modules. Besides DNA and RNAs, i con ains POL-I, UBF, FBL, and
TOPOI [145].
Among he d ugs supp essing he la e s eps o he RNA p ocessing
Table 1 (con inued)
S ess ype Cause E ec on nucleola
s uc u e
Re .
Se /Th p o ein
phospha ase
inhibi ion
Okadaic acid Nucleola accumula ion
o p80 coilin and splicing
small nuclea
ibonucleop o eins
(snRNPs)
[151]
Vi al in ec ion Adeno i us,
co ona i us,
polio i us, e c.
Changes in nucleola
mo phology and
composi ion
[152,153]
Nu ien
dep i a ion
Se um emo al Down egula ion o DNA
ansc ip ion and
educed ibosome
p oduc ion
[154]
Hypoxia Reduc ion o he
cellula concen a ion
o O
2
Nucleola dis up ion,
down egula ion o DNA
ansc ip ion
[155]
Oxida i e s ess H
2
O
2
T ansloca ion o
nucleola p o eins
(NPM) o nucleoplasm,
down egula ion o DNA
ansc ip ion
[156]
Osmo ic s ess Hypoosmo ic
condi ions
No de e mined [157]
Osmo ic s ess Hype osmo ic
condi ions
Loss o nucleola FC,
ansloca ion o
nucleola p o eins (NCL)
o nucleoplasm
[158]
The mal s ess Hea shock Accumula ion o
mis olded p o eins in he
GC
[159]
Fo ma ion o A-bodies [109,110]
Cold-wa m shock Expo o he RNA
exosome complex om
he nucleoli o he
nucleoplasm
[160]
Acidosis pH dec ease Fo ma ion o A-bodies [109,110]
K. Gonz´
alez-A zola
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
9
a e molecules classically ela ed o DNA me abolism, bu hey also a ec
RNA me abolism. This is he case o he ype II opoisome ase poison
e oposide and he DNA/RNA syn hesis inhibi o 5- luo ou acil [132].
The p o easome inhibi o s MG-132 and bo ezomib, besides he ans-
la ion inhibi o s homoha ing onine and cycloheximide, also block la e
RNA p ocessing [132] by unde e mined mechanisms. Rema kably,
compounds inhibi ing la e RNA p ocessing appa en ly did no induce
al e a ions in nucleola s uc u e [132].
A de i a i e o a h ombin-binding ap ame (TBA), which is a sho
DNA oligonucleo ide ha adop s G-quad uplex s uc u es, has been e-
po ed o induce nucleola s ess by impai ing RNA p ocessing, leading
o he accumula ion o p e- RNAs. This causes cell cycle a es o
apop osis in p53-de icien colon cance cells [146]. Rema kably, he
cy o oxic ac i i y o he TBA de i a i e is media ed by he ibosomal
p o ein RPL3, which is ansloca ed om he nucleolus o he nucleo-
plasm o posi i ely egula e p21 exp ession in a p53-independen
manne [146]. Owing o i s an ip oli e a i e ac i i y, his ap ame can
be used as a he apeu ic ool agains umo de elopmen due o i s
Fig. 4. Schema ic ep esen a ion o ac ion mechanisms o compounds a ge ing ibosome biogenesis.
O e iew o he e ec s o di e se an icance d ugs on DNA ansc ip ion, ea ly o la e RNA p ocessing, ibosomal subuni assembly and ma u a ion, and nuclea
expo o he ibonucleop o ein complexes. P ocessing he 47S RNA p ecu so in o ma u e 28S, 5.8S, and 18S RNAs is a mul i-s ep p ocess ha p oduces di e en
RNA in e media es (e.g., 32S RNA). Ac inomycin D (low concen a ion), mi omycin C, me ho exa e, BMH-21, me a es in, and PMR-116 block ansc ip ion by
RNA polyme ase-I (POL-I). CX-5461 dis up s he in e ac ion be ween p omo e selec i i y ac o complex (SL1) and POL-I, whe eas ellip icines elici SL1 dissocia ion
om he DNA p omo e . Sodium ce i as a in and apamycin in e e e wi h he ansc ip ion ini ia ion ac o -IA (TIF-IA)-POL-I in e ac ion. Doxo ubicin and
mi oxan one abolish DNA ansc ip ion by inhibi ing ype-II opoisome ases (TOPO II). Oxalipla in and cispla in (a high doses) o m adduc s wi h DNA and
ups eam binding ac o (UBF), leading o UBF seques a ion. BMH-21, aminoac idines, he nandonine, and sempe i ine p omo e he ubiqui in-media ed p o easome
deg ada ion o one ca aly ic subuni o POL-I (RPA194). CX-3543 in e up s he binding o DNA G-quad uplex o nucleolin (NCL). T ip olide dis up s he o ma ion
o he ansc ip ion ac o -IIIB (TFIIIB) complex a he 5S DNA p omo e , hus down egula ing RNA polyme ase III (POL-III). Camp o hecin and i s de i a i es a e
ype-I opoisome ase inhibi o s ha mainly a ec ea ly p ocessing o he RNA. Aminoac idines also in e e e wi h his ea ly p ocess. Fla opi idol and osco i ine a e
cyclin-dependen kinase-9 (CDK9) inhibi o s ha impede phospho yla ion and ac i a ion o RNA polyme ase-II (POL-II), hus a oiding he POL-II-media ed an-
sc ip ion o he small nucleola RNA (snoRNA) U8, essen ial o ea ly RNA p ocessing. CDK9 inhibi o s also p omo e he POL-I-in e posed DNA ansc ip ion by
hampe ing he POL-II-media ed ansc ip ion o an i-sense in e genic non-coding RNAs (asincRNAs) ha coun e ac he ad e se e ec s o POL-I- ansc ibed sense
in e genic non-coding RNAs (sincRNAs). 5,6-dichlo o-1-be a-D- ibo u anosylbenzimidazole (DRB) is a casein kinase II (CKII) inhibi o ha p e en s ac i a ion o
POL-II, which, in u n, in e e es wi h he ea ly p ocessing o RNA by snoRNA U8. E oposide, MG-132, bo ezomib, homoha ing onine, cycloheximide, and 5- luo-
ou acil hinde he la e s eps o he RNA p ocessing p ocess by unde e mined mechanisms: diazabo ine and ibozinoindole-1 block assembly and ma u a ion o he
la ge ibosomal subuni , espec i ely. Lep omycin B hinde s he expo ecep o ch omosome egion main enance 1 (CRM1), hus inhibi ing he nuclea expo o
ibosomal subuni s. P: a phospho yla ed amino-acid esidue.
K. Gonz´
alez-A zola
BBA - Gene Regula o y Mechanisms 1867 (2024) 195029
16
Acknowledgemen s
The au ho would like o exp ess g a i ude o D . Jose Ca los Reyes
o p o iding cons uc i e c i icism on he e iew.
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