Nucleolus

the

fascinating

nuclear

body

Nucleoli

are

the

prominent

contrasted

structures

of

the

cell

nucleus

In

the

nucleolus

ribosomal

RNAs

are

synthesized

processed

and

assembled

with

ribosomal

proteins

RNA

polymerase

I

synthesizes

the

ribosomal

RNAs

and

this

activity

is

cell

cycle

regulated

The

nucleolus

reveals

the

functional

organization

of

the

nucleus

in

which

the

compartmentation

of

the

different

steps

of

ribosome

biogenesis

is

observed

whereas

the

nucleolar

machineries

are

in

permanent

exchange

with

the

nucleoplasm

and

other

nuclear

bodies

After

mitosis

nucleolar

assembly

is

a

time

and

space

regulated

process

controlled

by

the

cell

cycle

In

addition

by

generating

a

large

volume

in

the

nucleus

with

apparently

no

RNA

polymerase

II

activity

the

nucleolus

creates

a

domain

of

retention/sequestration

of

molecules

normally

active

outside

the

nucleolus

Viruses

interact

with

the

nucleolus

and

recruit

nucleolar

proteins

to

facilitate

virus

replication

The

nucleolus

is

also

a

sensor

of

stress

due

to

the

redistribution

of

the

ribosomal

proteins

in

the

nucleoplasm

by

nucleolus

disruption

The

nucleolus

plays

several

crucial

functions

in

the

nucleus

in

addition

to

its

function

as

ribosome

factory

of

the

cells

it

is

a

multifunctional

nuclear

domain

and

nucleolar

activity

is

linked

with

several

pathologies

Perspectives

on

the

evolution

of

this

research

area

are

proposed

Introduction

Brief

history

of

the

nucleolus

An

ovoid

body

visible

in

the

nucleus

was

probably

the

first

observation

of

the

nucleolus

more

than

two

centuries

ago

by

F.

Fontana

Since

that

time

the

nucleolus

has

been

the

object

of

intense

investigation

and

interestingly

our

vision

of

the

nucleolus

has

evolved

with

technical

progress

During

the

nineteenth

century

using

light

microscopy

numerous

cytologists

described

the

variability

of

nucleolar

morphology

with

great

precision

-LRB-

Montgomery

-RRB-

In

McClintock

proposed

that

the

nucleolus

is

organized

in

the

telophase

through

the

activity

of

the

nucleolar-organizing

body

-LRB-

McClintock

-RRB-

Since

the

nucleolar-organizing

body

corresponds

to

a

specific

region

of

chromosome

in

Zea

mays

this

was

the

first

time

the

nucleolus

was

related

to

gene

activity

In

the

's

the

presence

of

RNAs

in

the

nucleolus

was

demonstrated

and

in

the

's

in

situ

hybridization

techniques

made

it

possible

to

identify

ribosomal

genes

-LRB-

rDNAs

-RRB-

in

the

nucleolar

organizer

region

-LRB-

NOR

-RRB-

-LRB-

Caspersson

Perry

Ritossa

and

Spiegelman

-RRB-

During

the

same

period

mass

isolation

of

nucleoli

became

possible

leading

to

the

biochemical

characterization

of

nucleolar

components

Based

on

these

results

it

was

proposed

that

ribosome

biogenesis

occurs

in

nucleoli

Given

that

the

nucleolus

became

a

subject

of

great

interest

the

International

symposium

on

the

nucleolus

its

structure

and

function

was

organized

in

Montevideo

in

and

the

contributions

published

in

Natl

Cancer

Inst

Monogr

no

-LRB-

USA

-RRB-

in

Since

at

the

initiative

of

W.

Bernhard

and

H.

Busch

Nucleolar

Workshops

on

nucleolar

organization

the

biochemistry

of

nucleolar

proteins

rRNA

processing

as

well

as

variability

in

cancer

cells

were

regularly

organized

Several

books

on

nucleoli

were

published

among

them

the

famous

The

nucleolus

and

ribosome

biogenesis

is

still

a

very

useful

source

of

information

-LRB-

Hadjiolov

-RRB-

Between

and

the

functional

organization

of

the

nucleolus

was

deciphered

in

large

part

due

to

the

improvement

of

labeling

by

the

electron

microscopy

-LRB-

EM

-RRB-

Recently

a

new

field

of

investigation

was

opened

when

molecules

not

involved

in

ribosome

biogenesis

were

detected

in

the

nucleolus

-LRB-

Carmo-Fonseca

et

al.

Pederson

Politz

et

al.

Visintin

and

Amon

-RRB-

In

accordance

with

these

nucleolar

localizations

nucleolar

mass

spectrometry

analyses

identified

nucleolar

proteins

some

of

them

not

related

to

ribosome

biogenesis

-LRB-

Andersen

et

al.

-RRB-

The

area

of

plurifunctional

nucleolus

was

opened

Consequently

The

nucleolus

a

book

that

presents

the

state

of

the

art

was

published

-LRB-

Olson

-RRB-

as

well

as

several

reviews

on

the

multiple

functions

of

the

nucleolus

-LRB-

Boisvert

et

al.

Hernandez-Verdun

Hiscox

Raska

et

al.

-RRB-

General

information

The

nucleolus

an

organelle

formed

by

the

act

of

building

a

ribosome

-LRB-

Mélèse

and

Xue

-RRB-

reveals

by

its

size

and

organization

the

efficiency

of

ribosome

biogenesis

For

example

the

nucleolus

is

a

prominent

nuclear

structure

in

cycling

cells

but

of

limited

size

in

the

terminal

stages

of

differentiation

such

as

in

lymphocytes

or

chick

erythrocytes

If

ribosome

biogenesis

is

blocked

reorganization

of

the

nucleolar

components

is

visible

in

segregated

nucleoli

In

mammalian

cells

the

nucleolus

is

disorganized

in

prophase

and

reassembled

at

the

end

of

mitosis

using

the

nucleolar

machineries

from

the

previous

cell

cycle

On

the

contrary

in

yeast

the

nucleolus

is

present

and

active

throughout

the

cell

cycle

even

though

condensation

of

the

rDNAs

is

necessary

for

transmission

of

the

nucleolus

in

anaphase

-LRB-

D'Amours

et

al.

Sullivan

et

al.

Torres-Rosell

et

al.

-RRB-

The

nucleolus

is

the

ribosome

factory

of

the

cell

In

the

nucleolus

rDNAs

are

transcribed

the

precursor

ribosomal

RNAs

-LRB-

pre-rRNAs

-RRB-

are

cleaved

processed

and

assembled

with

the

ribosomal

proteins

and

the

RNA

to

form

the

and

ribosomal

subunits

-LRB-

selected

reviews

Gébrane-Younès

et

al.

Hernandez-Verdun

and

Junéra

Scheer

et

al.

Scheer

and

Hock

Shaw

and

Jordan

Thiry

and

Goessens

-RRB-

This

complex

series

of

maturation

and

processing

events

presently

better

characterized

in

yeast

than

in

higher

eukaryotes

is

under

the

control

of

about

small

nucleolar

RNAs

-LRB-

snoRNAs

-RRB-

and

large

RNP

complexes

-LRB-

-RRB-

the

small

subunit

-LRB-

SSU

-RRB-

processome

containing

the

snoRNAs

and

proteins

or

Utps

-LRB-

U

three

proteins

-RRB-

required

for

the

ribosomal

subunit

and

-LRB-

-RRB-

the

large

subunit

-LRB-

LSU

-RRB-

processome

required

for

the

ribosomal

subunit

-LRB-

de

la

Cruz

et

al.

Fatica

and

Tollervey

Fromont-Racine

et

al.

Sollner-Webb

et

al.

Tollervey

-RRB-

The

snoRNAs

associated

with

proteins

function

in

the

maturation

of

rRNAs

creating

two

types

of

modified

nucleotides

-LRB-

O-methylation

and

pseudouridylation

-RRB-

and

mediating

endonucleolytic

cleavages

of

pre-rRNAs

-LRB-

Gerbi

and

Borovjagin

-RRB-

Our

objective

is

to

focus

this

review

on

the

ribosome

biogenesis

processes

occurring

in

the

nucleoli

that

might

help

to

decipher

the

global

organization

of

nuclear

functions

We

describe

nucleolar

organization

and

dynamics

propose

our

view

on

nucleolar

targeting

report

the

relationship

between

the

nucleolus

and

the

cell

cycle

review

particular

relationships

between

nucleolus

and

virus

and

nucleolus

related

to

cancer

The

nucleolus

is

a

model

of

compartmentation

Three

main

components

in

the

active

nucleolus

The

nucleolus

has

been

proposed

as

the

paradigm

of

nuclear

functional

compartmentalization

-LRB-

Strouboulis

and

Wolffe

-RRB-

It

is

the

site

of

ribosome

biogenesis

and

in

addition

the

nucleolar

machineries

are

distributed

in

different

compartments

When

observed

by

EM

three

main

nucleolar

components

-LRB-

compartments

-RRB-

can

be

discerned

in

mammalian

cells

the

fibrillar

centers

-LRB-

FCs

-RRB-

the

dense

fibrillar

component

-LRB-

DFC

-RRB-

and

the

granular

component

-LRB-

GC

-RRB-

-LRB-

Fig.

-RRB-

The

FCs

are

clear

areas

partly

or

entirely

surrounded

by

a

highly

contrasted

region

-LRB-

Goessens

et

al.

-RRB-

the

DFC

The

FCs

and

the

DFC

are

embedded

in

the

GC

mainly

composed

of

granules

of

nm

in

diameter

The

most

contrasted

structures

in

the

EM

sections

stained

with

uranyl

and

lead

correspond

to

high

concentrations

of

nucleic

acids

The

condensed

chromatin

surrounding

part

of

the

nucleolus

is

visible

using

standard

or

preferential

staining

methods

and

also

as

a

network

within

the

nucleolus

-LRB-

Fig.

-RRB-

The

global

amount

of

intra-nucleolar

chromatin

is

probably

low

since

by

light

microscopy

DNA

staining

by

DAPI

excludes

the

nucleolus

Fig.

nucleolus

of

mammalian

cells

as

seen

by

electron

microscopy

a

In

the

human

HeLa

cell

the

three

main

nucleolar

components

are

visible

in

a

section

of

material

fixed

in

glutaraldehyde

and

osmium

tetroxyde

embedded

in

Epon

and

the

section

contrasted

with

uranyl

acetate

and

lead

citrate

FCs

of

different

sizes

are

visible

and

the

largest

is

indicated

by

an

asterisk

The

FCs

are

surrounded

by

the

DFC

and

are

embedded

in

the

GC

b

Preferential

contrast

of

DNA

using

NAMA-Ur

staining

in

a

cell

-LRB-

courtesy

J.

Gébrane-Younès

-RRB-

The

nucleolus

is

the

gray

structure

surrounded

by

highly

contrasted

chromatin

-LRB-

arrow

-RRB-

Some

chromatin

filaments

are

also

visible

inside

of

the

nucleolus

-LRB-

Nu

-RRB-

c

d

Nucleolus

of

rat

neurones

-LRB-

courtesy

M.

J.

Pébusque

-RRB-

in

the

day

-LRB-

c

-RRB-

and

during

the

night

-LRB-

d

-RRB-

which

is

the

active

period

for

the

nucleolus

of

the

rat

In

the

nonactive

period

-LRB-

c

-RRB-

the

nucleolus

is

reticulated

with

small

FCs

-LRB-

asterisk

-RRB-

In

the

active

period

one

giant

FC

is

visible

-LRB-

d

asterisk

-RRB-

Bar

in

a

μm

and

bars

in

b

c

and

d

μm

It

has

become

apparent

that

nucleoli

of

different

cell

types

exhibit

a

variable

number

of

FCs

of

different

sizes

with

an

inverse

proportion

between

size

and

number

-LRB-

Hozak

et

al.

Pébusque

and

Seïte

-RRB-

Generally

cells

with

a

high

rate

of

ribosome

biogenesis

possess

numerous

small

FCs

On

the

contrary

cells

with

greatly

reduced

metabolic

and

transcription

activities

present

small

nucleoli

with

one

large-sized

FC

such

as

in

lymphocytes

and

in

inactive

mammalian

neurons

-LRB-

Hozàk

et

al.

Lafarga

et

al.

-RRB-

In

the

more

active

neurons

one

giant

FC

-LRB-

GFC

-RRB-

of

μm

is

observed

together

with

small

FCs

-LRB-

Fig.

d

-RRB-

It

was

demonstrated

that

the

GFC

is

enriched

in

the

upstream

binding

factor

the

UBF

transcription

factor

in

a

small

ubiquitin-like

modifier

-LRB-

SUMO

-RRB-

and

but

lack

ubiquitin-proteasome

and

proteasome

-LRB-

Casafont

et

al.

-RRB-

However

the

possibility

that

only

one

FC

might

play

a

role

in

storage

and

become

a

GFC

during

intense

nucleolar

activity

is

still

an

open

question

It

is

also

remarkable

that

the

tripartite

nucleolar

organization

is

not

general

since

the

nucleoli

of

Drosophila

and

insects

lack

FCs

-LRB-

Knibiehler

et

al.

Knibiehler

et

al.

-RRB-

It

has

been

proposed

that

this

difference

in

organization

could

be

linked

to

the

evolution

of

the

rDNAs

in

particular

to

the

size

of

the

intergenic

sequences

-LRB-

Thiry

and

Lafontaine

-RRB-

The

localization

of

the

nucleolar

machineries

is

related

to

their

function

in

the

production

of

the

small

and

large

ribosome

subunits

These

findings

have

led

to

assigning

specific

functions

to

specific

compartments

of

the

nucleolus

Nascent

transcripts

appear

at

the

junction

between

the

FCs

and

DFC

and

accumulate

in

the

DFC

-LRB-

Cmarko

et

al.

Guillot

et

al.

Hozàk

et

al.

Puvion-Dutilleul

et

al.

Shaw

and

Jordan

-RRB-

This

was

recently

confirmed

in

the

GFC

since

no

transcripts

can

be

detected

in

these

large

structures

-LRB-

Casafont

et

al.

-RRB-

Processing

of

the

pre-rRNA

starts

at

the

site

of

transcription

in

the

DFC

-LRB-

Cmarko

et

al.

-RRB-

and

continues

during

the

intra-nucleolar

migration

of

the

RNA

towards

the

GC

The

nucleolar

proteins

that

participate

in

the

early

stages

of

rRNA

processing

localize

in

the

DFC

such

as

fibrillarin

and

nucleolin

along

with

the

snoRNAs

-LRB-

Biggiogera

et

al.

Ginisty

et

al.

Ochs

et

al.

Puvion-Dutilleul

et

al.

-RRB-

whereas

proteins

-LRB-

nucleophosmin

-RRB-

and

PM-Scl

-LRB-

in

yeast

-RRB-

that

are

involved

in

intermediate

or

later

stages

of

processing

have

been

localized

to

the

GC

-LRB-

Biggiogera

et

al.

Gautier

et

al.

-RRB-

Recent

advances

in

the

isolation

of

large

RNP

complexes

by

tandem

affinity

purification

and

the

characterization

of

their

constituents

demonstrated

that

two

largely

independent

processing

machineries

exist

in

yeast

nucleoli

the

SSU

processome

-LRB-

Dragon

et

al.

Grandi

et

al.

-RRB-

and

the

LSU

processing/assembly

factors

-LRB-

Raué

-RRB-

The

processome

is

localized

in

the

DFC

and

most

of

the

processing

occurs

in

the

GC

There

is

no

particular

domain

characterized

in

the

GC

corresponding

to

the

subunit

This

is

most

probably

due

to

the

limited

events

of

processing

in

the

GC

since

the

last

step

of

processing

occurs

in

the

cytoplasm

In

conclusion

it

seems

that

in

the

nucleoli

the

vectorial

distribution

of

the

machineries

successively

involved

in

ribosome

biogenesis

correlates

with

the

different

processing

steps

of

the

biogenesis

of

the

ribosome

subunits

When

ribosome

biogenesis

is

active

the

confinement

of

certain

machineries

in

the

FCs

DFC

or

GC

makes

it

possible

to

reveal

these

subnucleolar

constituents

by

immunofluorescence

as

illustrated

for

FCs

-LRB-

Fig.

-RRB-

DFC

-LRB-

Fig.

b

-RRB-

and

GC

-LRB-

Fig.

d

-RRB-

The

factors

associated

with

the

rDNA

transcription

machinery

are

distributed

in

several

foci

most

frequently

inside

the

nucleolar

volume

as

illustrated

for

UBF

These

foci

correspond

to

FCs

A

distribution

within

the

network

inside

the

nucleolus

is

typical

of

the

DFC

as

demonstrated

for

fibrillarin

Labeling

of

the

nucleolar

volume

excluding

small

areas

contained

within

the

volume

is

typical

of

the

GC

as

illustrated

for

These

labeling

patterns

-LRB-

FCs

DFC

GC

-RRB-

in

the

nucleoli

provide

a

good

indication

of

the

step

of

ribosome

biogenesis

concerned

and

also

reveal

the

blockage

of

ribosome

biogenesis

when

this

organization

is

disturbed

-LRB-

see

below

-RRB-

Fig.

subnucleolar

constituents

revealed

by

fluorescence

microscopy

-LRB-

A

-RRB-

The

rDNA

transcription

machinery

illustrated

by

UBF

labeling

is

localized

in

several

foci

corresponding

to

FCs

in

HeLa

cells

-LRB-

a

-RRB-

rDNA

transcription

sites

detected

by

in

situ

BrUTP

incorporation

-LRB-

b

-RRB-

mainly

colocalize

with

UBF

as

seen

by

the

merge

-LRB-

c

-RRB-

The

nucleus

is

visualized

by

Dapi

staining

-LRB-

d

-RRB-

-LRB-

B

-RRB-

HeLa

cells

expressing

either

fibrillarin-GFP

fusion

or

fusion

Fibrillarin

decorates

the

DFC

-LRB-

a

b

-RRB-

whereas

decorates

the

GC

-LRB-

c

d

-RRB-

In

ActD-treated

cells

nucleoli

are

segregated

fibrillarin

localizes

in

caps

-LRB-

e

f

-RRB-

contrary

to

that

localizes

in

the

central

body

and

outside

the

caps

-LRB-

g

h

-RRB-

Arrowheads

point

the

caps

Bars

μm

Signature

of

impaired

ribosome

biogenesis

Transient

association

of

functionally

related

components

appears

necessary

to

generate

a

morphologically

defined

nucleolus

with

its

three

distinct

components

thereby

maintaining

the

nucleolus

in

its

usual

organization

This

suggests

that

such

an

organization

results

from

the

activity

of

ribosome

biogenesis

Indeed

nucleolar

reorganization

is

induced

when

ribosome

biogenesis

is

impaired

either

by

inhibiting

rDNA

transcription

or

inhibiting

rRNA

processing

and/or

transport

Inhibition

of

transcription

Nucleolar

segregation

is

observed

upon

rDNA

transcriptional

arrest

either

in

physiological

conditions

or

induced

by

low

doses

of

actinomycin

D

-LRB-

ActD

-RRB-

The

segregation

of

nucleoli

is

characterized

by

the

separation

of

the

nucleolar

components

that

remain

close

to

each

other

but

no

longer

intermingle

-LRB-

Fig.

h

-RRB-

-LRB-

for

reviews

see

Hadjiolov

Hernandez-Verdun

and

Junéra

Scheer

and

Benavente

-RRB-

The

effect

of

ActD

on

nucleolar

organization

follows

sequential

changes

first

the

fibrillar

components

-LRB-

FCs

and

DFC

-RRB-

condense

and

migrate

towards

the

periphery

of

the

nucleolus

after

which

the

nucleolar

components

segregate

to

finally

form

a

central

body

associated

with

caps

-LRB-

Hadjiolova

et

al.

-RRB-

In

the

caps

are

several

proteins

related

to

the

RNA

polymerase

-LRB-

pol

-RRB-

I

transcription

machinery

such

as

UBF

close

to

fibrillarin-containing

caps

In

the

central

body

are

proteins

derived

from

the

GC

some

of

which

are

progressively

released

such

as

PM-Scl

It

was

recently

demonstrated

that

certain

nucleolar

caps

of

segregated

nucleoli

could

recruit

factors

involved

in

mRNA

splicing

In

this

case

localization

is

induced

by

inhibition

of

both

RNA

pol

I

-LRB-

rRNA

transcription

arrest

-RRB-

and

RNA

pol

II

-LRB-

mRNA

transcription

arrest

-RRB-

-LRB-

Shav-Tal

et

al.

-RRB-

This

is

not

observed

when

only

RNA

pol

I

is

inhibited

indicating

that

the

composition

of

a

segregated

nucleolus

can

be

more

complex

when

induced

by

general

transcription

inhibition

One

question

that

remains

unanswered

is

how

nucleolar

components

continue

to

be

maintained

in

segregated

nucleoli

in

spite

of

the

absence

of

transcription

or

pre-rRNA

processing

Nucleolar

proteins

may

still

be

capable

of

forming

complexes

during

inhibition

of

transcription

but

why

these

complexes

remain

juxtaposed

is

presently

unknown

Recently

it

was

reported

that

re-localization

of

proteins

in

specific

caps

of

segregated

nucleoli

-LRB-

after

inhibition

of

RNA

pol

I

and

II

transcription

-RRB-

is

an

energy-dependent

repositioning

process

that

requires

active

metabolism

of

the

cells

-LRB-

Shav-Tal

et

al.

-RRB-

most

probably

also

ATP

and

GTP

Degradation

of

rRNAs

A

clue

to

the

question

of

rRNA

degradation

in

the

nucleolus

was

recently

proposed

in

yeast

a

surveillance

pathway

that

eliminates

defective

pre-ribosomal

subunits

after

addition

of

poly

-LRB-

A

-RRB-

tails

was

described

-LRB-

LaCava

et

al.

-RRB-

RNA

degradation

appears

to

occur

preferentially

within

a

subnucleolar

structure

the

No-body

and

is

mediated

by

the

exosome

-LRB-

Dez

et

al.

-RRB-

Similarly

when

the

nuclear

protein

of

the

exosome

was

deleted

poly

-LRB-

A

-RRB-

rRNAs

and

poly

-LRB-

A

-RRB-

snoRNAs

colocalized

in

one

focus

with

-LRB-

fibrillarin

in

human

-RRB-

most

probably

the

No-body

-LRB-

Carneiro

et

al.

-RRB-

This

body

is

distinct

from

the

nucleolar

body

that

functions

in

snoRNA

maturation

in

yeast

and

could

be

a

compartment

where

polyadenylation

and

degradation

of

nucleolar

RNAs

take

place

This

compartmentation

would

promote

efficient

recognition

of

rRNAs

in

view

of

degradation

by

the

exosome

-LRB-

Carneiro

et

al.

-RRB-

During

nucleolar

segregation

induced

by

ActD

in

human

cells

rRNAs

are

degraded

However

the

formation

of

one

focus

containing

PM-Scl

has

not

been

described

it

could

be

either

an

early

event

that

was

not

carefully

examined

or

rRNA

degradation

could

be

different

in

yeast

and

mammalian

cells

Disconnection

of

the

nucleolar

component

Disconnection

between

rDNA

transcription

sites

and

the

late

rRNA

processing

proteins

can

be

induced

either

by

kinase

inhibitors

or

by

modifications

of

snoRNA

domains

-LRB-

Chan

et

al.

Colau

et

al.

David-Pfeuty

et

al.

Rubbi

and

Milner

Sirri

et

al.

-RRB-

The

separation

of

the

DFC

and

GC

can

be

reversed

by

removal

of

a

inhibitor

restoring

nucleolar

organization

The

kinase

is

known

to

phosphorylate

several

nucleolar

proteins

-LRB-

Meggio

and

Pinna

-RRB-

We

postulate

that

the

connection

between

DFC

and

GC

is

controlled

at

least

in

part

by

phosphorylation

of

these

proteins

This

hypothesis

was

verified

for

by

mutation

of

the

major

site

of

phosphorylation

-LRB-

Louvet

et

al.

-RRB-

In

conclusion

the

rRNA

processing

proteins

can

be

disconnected

from

the

rRNA

transcription

sites

indicating

that

rRNA

transcripts

are

not

sufficient

to

attract

the

processing

proteins

in

these

conditions

The

dynamics

of

nucleolar

reformation

and

the

connection

between

DFC

and

GC

is

ATP/GTP

dependent

sensitive

to

temperature

and

is

Traffic

and

dynamics

of

nucleolar

actors

The

analysis

in

living

cells

of

intranuclear

dynamics

has

recently

become

possible

using

fluorescent

fusion

proteins

Time-lapse

videomicroscopy

can

track

the

movement

of

large

fluorescent

complexes

in

the

cell

and

fluorescent

recovery

after

photobleaching

-LRB-

FRAP

-RRB-

can

measure

the

intracellular

mobility

or

the

residency

time

of

fluorescent

proteins

-LRB-

Lippincott-Schwartz

et

al.

-RRB-

Inverse

FRAP

-LRB-

iFRAP

-RRB-

quantifies

the

loss

of

fluorescence

of

the

region

of

interest

-LRB-

ROI

-RRB-

after

complete

bleaching

outside

this

region

-LRB-

Dundr

et

al.

-RRB-

This

constitutes

a

direct

evaluation

of

the

residency

time

of

the

proteins

in

the

ROI

Another

approach

is

the

use

of

photoactivatable

GFP

-LRB-

PA-GFP

-RRB-

to

follow

the

traffic

of

the

activated

proteins

-LRB-

Patterson

and

Lippincott-Schwartz

-RRB-

This

process

is

similar

to

pulse-chase

experiments

since

it

makes

it

possible

to

follow

a

pool

of

labeled

proteins

starting

at

time

zero

These

technologies

applied

to

nuclear

dynamics

have

introduced

new

dimensions

and

unexpected

concepts

concerning

nuclear

functional

compartmentation

The

mobility

of

several

GFP-tagged

nuclear

proteins

-LRB-

nucleolar

proteins

histones

DNA

binding

proteins

transcription

factors

splicing

factors

nuclear

receptors

-RRB-

has

been

estimated

by

FRAP

and

the

recovery

of

fluorescence

was

slower

than

predicted

for

isolated

diffusing

proteins

of

similar

size

FRAP

recovery

rates

change

with

inhibition

of

transcription

decreased

temperature

and

depletion

of

ATP

indicating

that

recovery

is

correlated

with

nuclear

activity

It

was

demonstrated

that

nucleolar

proteins

rapidly

associate

with

and

dissociate

from

nucleolar

components

in

continuous

exchanges

with

the

nucleoplasm

-LRB-

Phair

and

Misteli

-RRB-

The

recovery

curve

of

GFP-fibrillarin

-LRB-

DFC

marker

-RRB-

in

the

nucleolus

reached

a

plateau

s

after

bleaching

and

the

plateau

indicated

an

immobile

fraction

of

The

diffusion

coefficient

of

fibrillarin

-LRB-

estimated

between

and

s

-RRB-

was

times

lower

in

the

nucleolus

than

in

the

nucleoplasm

-LRB-

Chen

and

Huang

Phair

and

Misteli

Snaar

et

al.

-RRB-

This

value

is

proposed

to

reflect

the

time

of

residency

of

fibrillarin

engaged

in

nucleolar

activity

and

could

explain

the

fact

that

the

time

of

residency

of

fibrillarin

is

shorter

in

the

Cajal

body

than

in

the

nucleolus

-LRB-

Dundr

et

al.

-RRB-

The

nucleolar

proteins

engaged

in

rRNA

transcription

and

processing

-LRB-

respectively

UBF

nucleolin

and

-RRB-

also

move

with

rapid

recovery

rates

in

the

nucleolus

as

does

fibrillarin

-LRB-

Chen

and

Huang

Louvet

et

al.

-RRB-

Conversely

the

recovery

rates

of

ribosomal

proteins

are

slow

-LRB-

times

slower

than

nucleolar

proteins

-RRB-

This

could

reflect

a

slower

mechanism

for

ribosome

protein

assembly

compared

with

transcription

and

processing

-LRB-

Chen

and

Huang

-RRB-

or

alternatively

more

stable

associations

of

ribosomal

proteins

with

the

pre-rRNAs

-LRB-

also

designated

NPM

-RRB-

is

a

multifunctional

protein

abundant

in

the

GC

of

the

nucleolus

that

undergoes

different

phosphorylation

events

during

the

cell

cycle

It

was

recently

demonstrated

by

FRAP

that

the

kinetics

of

depends

on

its

phosphorylation

status

-LRB-

Negi

and

Olson

-RRB-

During

interphase

the

half-time

-LRB-

-RRB-

recovery

of

is

s

in

nucleoli

but

when

the

phosphorylation

site

is

mutated

-LRB-

-RRB-

the

increases

to

s

and

when

a

mutant

mimicking

phosphorylation

charges

of

the

four

sites

of

mitotic

phosphorylation

the

decreases

to

s

This

could

indicate

that

the

protein

has

a

higher

affinity

for

the

nucleolar

components

-LRB-

Negi

and

Olson

-RRB-

Alternatively

this

could

correspond

to

a

decreased

turner-over

in

the

nucleolar

complexes

in

correlation

with

the

disconnection

of

the

DFC

and

GC

occurring

by

overexpression

of

-LRB-

Louvet

et

al.

-RRB-

Overexpression

during

interphase

of

mimicking

four

sites

of

mitotic

phosphorylation

increased

the

mobility

of

It

is

tempting

to

propose

that

this

results

from

a

defect

of

affinity

for

rRNAs

of

these

as

demonstrated

for

mitotic

phosphorylation

of

-LRB-

Okuwaki

et

al.

-RRB-

Inhibition

of

pol

I

transcription

by

ActD

does

not

prevent

traffic

of

nucleolar

proteins

However

if

the

diffusion

coefficient

of

nucleolar

proteins

in

the

nucleoplasm

is

similar

for

active

and

repressed

pol

I

transcription

the

traffic

in

segregated

nucleoli

changes

differently

for

different

nucleolar

components

Traffic

of

UBF

in

the

nucleolus

is

decreased

by

ActD

whereas

it

is

similar

for

nucleolin

or

increased

for

ribosomal

proteins

-LRB-

Chen

and

Huang

-RRB-

In

contrast

to

the

well-defined

nucleolar

structures

visible

by

EM

all

the

nucleolar

proteins

involved

in

ribosome

biogenesis

that

have

been

examined

cycle

between

the

nucleolus

and

the

nucleoplasm

in

interphase

cells

To

summarize

it

is

now

established

that

rapid

diffusion

of

nucleolar

proteins

occurs

in

the

nucleoplasm

and

recruitment

to

the

nucleolus

is

permanent

Moreover

the

difference

in

kinetics

of

several

proteins

shared

between

the

nucleolus

and

the

Cajal

body

suggests

the

existence

of

compartment-specific

retention

-LRB-

Dundr

et

al.

-RRB-

Targeting

to

the

nucleolus

To

be

localized

or

retained

within

the

nucleolus

In

eukaryotic

cells

once

imported

or

diffused

into

the

nucleus

some

proteins

distribute

throughout

the

nucleoplasm

and

others

are

targeted

to

specific

nuclear

compartments

such

as

nucleoli

Proteomic

analyses

revealed

that

at

least

proteins

are

localized

in

nucleoli

-LRB-

Andersen

et

al.

Leung

et

al.

-RRB-

Whereas

the

rules

and

signals

that

govern

the

nuclear

localization

and

nuclear

export

of

proteins

are

now

well

defined

those

concerning

nucleolar

localization

are

still

debated

Contrary

to

the

nuclear

localization

signals

-LRB-

NLSs

-RRB-

nucleolar

localization

signals

or

sequences

-LRB-

NoLSs

-RRB-

are

not

well

characterized

Although

several

NoLSs

have

been

described

no

obvious

consensus

sequence

has

emerged

Nevertheless

all

NoLSs

reported

for

nucleolus

localizing

virus

proteins

such

as

Rev

-LRB-

Kubota

et

al.

-RRB-

Tat

-LRB-

Dang

and

Lee

-RRB-

and

human

T-cell

leukaemia

virus

type

Rex

-LRB-

Rex

-RRB-

-LRB-

Siomi

et

al.

-RRB-

and

for

cellular

proteins

such

as

the

nucleolar

protein

-LRB-

Valdez

et

al.

-RRB-

-LRB-

Song

and

Wu

-RRB-

and

-LRB-

Dang

and

Lee

-RRB-

are

rich

in

basic

residues

The

capacity

of

numerous

proteins

to

adopt

nucleolar

localization

has

been

correlated

with

interaction

of

these

proteins

with

Owing

to

the

ability

of

numerous

nucleolar

proteins

to

interact

with

and

because

this

major

nucleolar

protein

shuttles

constantly

between

the

nucleus

and

the

cytoplasm

-LRB-

Borer

et

al.

-RRB-

it

was

frequently

suggested

that

might

be

a

transporter

for

nucleolar

proteins

possessing

a

NoLS

-LRB-

Fankhauser

et

al.

Li

Valdez

et

al.

-RRB-

Even

if

this

tempting

hypothesis

has

never

been

demonstrated

recent

results

obtained

using

stable

cell

lines

with

reduced

expression

levels

showed

that

the

nucleolar

localization

of

ARF

is

linked

to

-LRB-

Korgaonkar

et

al.

-RRB-

Indeed

reduced

expression

of

induced

a

partial

delocalization

of

ARF

from

nucleoli

to

nucleoplasm

The

authors

therefore

concluded

that

targets

ARF

to

nucleoli

in

a

dose-dependent

manner

Nevertheless

this

result

does

not

allow

discriminating

between

a

role

for

in

the

transport

of

ARF

from

nucleoplasm

to

nucleoli

and/or

in

the

retention

of

ARF

in

nucleoli

A

NoLS

i.e.

a

sequence

essential

for

nucleolar

localization

is

most

probably

a

sequence

involved

in

nucleolar

retention

by

interacting

with

a

nucleolar

molecule

such

as

-LRB-

Lechertier

et

al.

-RRB-

Indeed

recent

analyses

of

the

intranuclear

dynamics

of

proteins

in

living

cells

revealed

that

nuclear

proteins

could

diffuse

within

the

nucleoplasm

-LRB-

Phair

and

Misteli

Sprague

and

McNally

-RRB-

As

for

the

nucleolus

it

was

demonstrated

that

nucleolar

proteins

rapidly

associate

with

and

dissociate

from

nucleolar

components

in

a

continuous

exchange

with

the

nucleoplasm

-LRB-

Chen

and

Huang

Dundr

et

al.

Phair

and

Misteli

Snaar

et

al.

-RRB-

There

probably

exist

compartment-specific

retention

mechanisms

for

proteins

in

nuclear

bodies

implying

that

the

residency

time

of

a

particular

molecule

in

a

given

nuclear

body

depends

on

its

specific

interactions

-LRB-

Misteli

-RRB-

In

support

of

this

possibility

we

have

recently

shown

that

the

fusion

of

a

sequence

with

fibrillarin

makes

it

possible

to

re-localize

fibrillarin

from

the

DFC

to

the

GC

of

nucleoli

where

is

mainly

localized

-LRB-

Lechertier

et

al.

-RRB-

Similarly

by

fusing

the

sequence

to

MafG

-LRB-

part

of

the

nuclear

transcription

factor

composed

of

both

MafG

and

-RRB-

is

redirected

from

the

nucleoplasm

to

the

GC

Therefore

interactions

most

probably

govern

the

nuclear

distribution

of

proteins

and

a

NoLS

is

very

likely

a

nucleolar

molecule-interacting

sequence

However

nucleolar

localization

of

a

protein

is

most

probably

governed

by

several

factors

and

the

presence

of

a

NoLS

in

its

sequence

is

not

sufficient

to

predict

nucleolar

localization

of

the

protein

In

particular

a

nucleolar

protein

must

first

be

localized

in

the

nucleus

and

consequently

all

mechanisms

that

interfere

with

nuclear

import

and/or

nuclear

export

of

a

nucleolar

protein

will

modify

its

localization

at

the

steady

state

A

good

illustration

is

provided

by

the

major

nucleolar

protein

This

multifunctional

protein

is

normally

mainly

located

in

the

GC

of

nucleoli

but

exhibits

an

aberrant

cytoplasmic

localization

in

one-third

of

acute

myeloid

leukemias

due

to

mutations

in

its

C-terminal

coding

exon

that

causes

a

frameshift

and

the

formation

of

an

additional

nuclear

export

signal

-LRB-

NES

-RRB-

-LRB-

Mariano

et

al.

-RRB-

Another

example

showing

the

difficulty

encountered

in

predicting

nucleolar

localization

is

provided

by

the

box

C/D

snoRNPs

it

seems

clear

that

the

nucleolar

localization

of

box

C/D

snoRNPs

is

linked

to

their

biogenesis

-LRB-

Verheggen

et

al.

Watkins

et

al.

-RRB-

Indeed

by

modifying

the

conserved

stem

II

of

the

box

C/D

motif

present

in

the

snoRNA

both

the

specific

assembly

of

the

box

C/D

snoRNP

and

nucleolar

localization

are

lost

-LRB-

Watkins

et

al.

-RRB-

Moreover

genetic

depletion

of

one

of

the

four

core

proteins

namely

kD

and

fibrillarin

also

inhibits

the

nucleolar

localization

of

box

C/D

snoRNPs

-LRB-

Verheggen

et

al.

-RRB-

However

targeting

of

box

C/D

snoRNPs

to

nucleoli

is

not

yet

fully

understood

Indeed

unexpectedly

two

nuclear

export

factors

PHAX

and

appear

to

be

stably

associated

with

the

pre-snoRNPs

-LRB-

Boulon

et

al.

Watkins

et

al.

-RRB-

Boulon

and

coworkers

proposed

that

precursors

bind

PHAX

which

targets

the

complex

to

the

Cajal

body

and

that

subsequently

further

targets

the

complexes

to

the

nucleolus

Even

if

PHAX

and

play

an

important

role

in

the

transport

of

box

C/D

snoRNPs

to

the

nucleus

the

possibility

that

these

proteins

may

also

function

in

the

nuclear

export

of

snoRNPs

can

not

be

excluded

-LRB-

Watkins

et

al.

-RRB-

This

possibility

is

reinforced

by

a

recent

study

showing

that

in

addition

to

nuclear

export

factors

the

nuclear

import

factor

Snurportin

is

involved

in

box

C/D

snoRNP

biogenesis

-LRB-

Watkins

et

al.

-RRB-

Nucleolar

localization

of

the

components

of

the

box

C/D

snoRNPs

would

therefore

depend

on

the

biogenesis

of

the

box

C/D

snoRNP

complexes

which

would

imply

nuclear

export

Control

of

rDNA

transcription

during

cell

cycle

rDNA

transcription

machinery

rDNAs

are

found

in

multiple

tandem

head-to-tail

arrayed

copies

in

the

nucleoli

of

eukaryotic

cells

-LRB-

Hadjiolov

-RRB-

In

mitotic

human

cells

rDNA

clusters

are

localized

on

the

short

arm

of

the

five

pairs

of

chromosomes

and

and

are

termed

NORs

Each

rDNA

unit

consists

of

a

transcribed

sequence

and

an

external

non-transcribed

spacer

-LRB-

Hadjiolov

Liau

and

Perry

-RRB-

in

which

all

the

sequences

necessary

for

proper

RNA

pol

I

transcription

such

as

proximal

promoters

spacer

promoters

and

terminators

are

located

-LRB-

Hadjiolov

-RRB-

In

the

rDNA

promoter

two

important

elements

have

been

described

a

CORE

element

and

an

upstream

control

element

-LRB-

UCE

-RRB-

-LRB-

Haltiner

et

al.

Windle

and

Sollner-Webb

Xie

et

al.

-RRB-

that

function

synergistically

to

recruit

a

transcriptionally

competent

RNA

pol

I

complex

This

complex

contains

in

addition

to

RNA

pol

I

the

upstream

binding

factor

-LRB-

UBF

-RRB-

-LRB-

Pikaard

et

al.

Voit

et

al.

-RRB-

the

selectivity

factor

protein

complex

-LRB-

Learned

et

al.

-RRB-

also

called

in

mouse

cells

-LRB-

Clos

et

al.

-RRB-

consisting

of

the

TATA-binding

protein

-LRB-

TBP

-RRB-

and

four

transcription

activating

factors

-LSB-

and

-LRB-

Comai

et

al.

Gorski

et

al.

Zomerdijk

et

al.

-RRB-

-RSB-

the

transcription

initiation

factor

TIF-IA

the

mouse

homolog

of

-LRB-

Bodem

et

al.

Moorefield

et

al.

-RRB-

and

the

transcription

termination

factor

-LRB-

Bartsch

et

al.

-RRB-

The

UBF

containing

HMG

boxes

-LRB-

Bachvarov

and

Moss

Jantzen

et

al.

-RRB-

that

confer

a

high

affinity

for

DNA

structures

plays

an

architectural

role

on

the

rDNA

promoter

-LRB-

Mais

et

al.

-RRB-

It

was

proposed

that

UBF

activates

rDNA

transcription

because

it

stabilizes

binding

of

required

to

recruit

the

initiation-competent

subfraction

of

RNA

pol

I

This

recruitment

is

achieved

by

interaction

of

UBF

with

the

RNA

pol

I-associated

factor

-LRB-

Schnapp

et

al.

-RRB-

and

by

interaction

of

with

-LRB-

Miller

et

al.

-RRB-

interacts

also

with

the

subunit

of

RNA

pol

I

and

thus

facilitates

linking

between

RNA

pol

I

and

complexes

-LRB-

Peyroche

et

al.

Yuan

et

al.

-RRB-

Following

initiation

is

released

and

can

associate

with

another

preinitiation

complex

Recycling

of

requires

a

post-translational

phosphorylation

event

that

appears

to

play

a

role

in

its

initiation

activity

-LRB-

Cavanaugh

et

al.

Zhao

et

al.

-RRB-

Moreover

it

was

proposed

that

is

not

only

involved

in

termination

of

transcription

in

cooperation

with

the

release

factor

PTRF

-LRB-

Jansa

and

Grummt

-RRB-

but

also

in

the

remodeling

of

ribosomal

chromatin

by

recruiting

ATP-dependent

remodeling

factors

to

the

rDNA

promoter

-LRB-

Längst

et

al.

-RRB-

The

nucleolar

remodeling

complex

-LRB-

NoRC

-RRB-

-LRB-

Strohner

et

al.

-RRB-

which

acts

in

repression

at

the

rDNA

promoter

level

-LRB-

Li

et

al.

Santoro

et

al.

-RRB-

and

the

transcription

activator

CSB

-LRB-

Cockayne

syndrome

group

B

protein

-RRB-

a

DNA-dependent

ATPase

interact

with

-LRB-

Yuan

et

al.

-RRB-

The

finding

that

interacts

with

both

CSB

and

NoRC

suggests

that

competitive

recruitment

of

CSB

and

NoRC

may

determine

the

epigenetic

state

of

the

rDNA

Regulation

during

the

cell

cycle

It

is

now

established

that

the

presence

of

a

fully

active

nucleolus

depends

on

cell

cycle

regulators

rDNA

transcription

is

maximum

in

the

S

and

phases

silent

in

mitosis

and

slowly

recovers

in

Post-translational

modifications

of

the

RNA

pol

I

machinery

are

required

for

the

formation

of

a

productive

preinitiation

complex

The

phosphorylation

status

of

several

components

of

the

RNA

pol

I

machinery

can

modify

the

activity

and

interactions

of

these

proteins

and

thus

can

modulate

rDNA

transcription

during

the

cell

cycle

Concerning

silencing

of

rDNA

transcription

during

mitosis

it

is

well

established

that

some

components

of

the

rDNA

transcription

machinery

such

as

-LRB-

Heix

et

al.

-RRB-

and

-LRB-

Sirri

et

al.

-RRB-

are

mitotically

phosphorylated

by

B

As

shown

in

vitro

B-mediated

phosphorylation

of

abrogates

its

transcriptional

activity

-LRB-

Heix

et

al.

-RRB-

Moreover

B

is

necessary

not

only

to

establish

repression

but

also

to

maintain

it

from

prophase

to

telophase

Indeed

in

vivo

inhibition

of

B

leads

to

dephosphorylation

of

the

mitotically

phosphorylated

forms

of

components

of

the

RNA

pol

I

machinery

and

restores

rDNA

transcription

in

mitotic

cells

-LRB-

Sirri

et

al.

-RRB-

On

the

other

hand

rDNA

transcription

also

appears

regulated

by

CDK

-LRB-

s

-RRB-

during

interphase

the

increase

of

rDNA

transcription

during

progression

depends

on

phosphorylation

of

UBF

by

CDK

cyclin

complexes

-LRB-

Voit

et

al.

-RRB-

and

CDK

inhibitor

treatments

partially

inhibit

rDNA

transcription

in

interphase

cells

-LRB-

Sirri

et

al.

-RRB-

Modifications

of

the

phosphorylation

status

of

UBF

and/or

affect

the

interactions

between

UBF

and

necessary

for

recruitment

of

RNA

pol

I

-LRB-

Zhai

and

Comai

-RRB-

In

addition

to

phosphorylation

it

has

been

speculated

that

acetyltransferases

might

also

regulate

the

activity

of

RNA

pol

I

transcription

factors

Indeed

two

studies

have

demonstrated

that

UBF

and

one

of

the

subunits

are

acetylated

in

vivo

-LRB-

Muth

et

al.

Pelletier

et

al.

-RRB-

Functional

studies

indicated

that

acetylated

UBF

is

transcriptionally

more

active

than

deacetylated

UBF

However

acetylation

of

UBF

does

not

affect

its

DNA

binding

activity

as

shown

for

other

transcription

factors

and

it

is

unclear

how

this

post-transcriptional

modification

modulates

UBF

activity

The

subunit

of

is

specifically

acetylated

by

recruitment

of

PCAF

-LRB-

associated

factor

-RRB-

to

the

rDNA

promoter

In

vitro

analyses

indicate

that

acetylation

of

is

likely

to

increase

the

activity

of

facilitating

interaction

of

the

complex

with

DNA

Sirtuins

the

human

homologues

of

the

yeast

-LRB-

silent

information

regulator

-RRB-

with

NAD-dependent

deacetylase

and

ADP-ribosyltranferase

activity

have

recently

been

implicated

in

the

regulation

of

the

RNA

pol

I

machinery

In

particular

nuclear

deacetylates

and

represses

RNA

pol

I

transcription

in

vitro

-LRB-

Muth

et

al.

-RRB-

Conversely

the

nucleolar

is

described

as

activator

of

rDNA

transcription

by

increasing

RNA

pol

I

recruitment

to

the

rDNA

but

no

substrates

of

such

activity

have

as

yet

been

identified

-LRB-

Ford

et

al.

-RRB-

Additional

in

vivo

approaches

are

necessary

to

better

understand

the

role

of

sirtuins

in

the

regulation

of

rDNA

transcription

SUMO

modification

is

reported

to

influence

the

assembly

of

transcription

factors

on

promoters

and

the

recruitment

of

chromatin-modifying

enzymes

and

is

often

associated

with

transcriptional

repression

-LRB-

Gill

-RRB-

Recently

the

colocalization

of

and

UBF

in

the

GFC

-LRB-

Casafont

et

al.

-RRB-

of

neuronal

cells

and

the

nucleolar

localization

of

the

sentrin/SUMO-specific

proteases

and

-LRB-

Gong

and

Yeh

Nishida

et

al.

-RRB-

suggest

a

potential

role

of

sumoylation

on

the

regulation

of

rDNA

transcription

Further

studies

of

the

identification

of

sumoylated

nucleolar

transcription

factors

will

be

necessary

to

verify

this

possibility

Nucleolar

assembly

and

disassembly

In

higher

eukaryotic

cells

at

the

beginning

of

mitosis

when

rDNA

transcription

is

repressed

the

nucleoli

disassemble

and

are

no

longer

observed

throughout

mitosis

Conversely

nucleoli

assemble

at

the

exit

from

mitosis

concomitantly

with

restoration

of

rDNA

transcription

and

are

functionally

active

throughout

interphase

Disassembly

in

prophase

In

late

prophase

when

mitotic

repression

of

rDNA

transcription

occurs

the

rDNA

transcription

machinery

remains

associated

with

rDNAs

in

the

NORs

as

revealed

by

the

analysis

of

different

components

at

the

steady

state

-LRB-

Roussel

et

al.

Sirri

et

al.

-RRB-

Nevertheless

more

recent

quantitative

kinetic

analyses

have

revealed

that

some

RNA

pol

I

subunits

including

and

transiently

dissociate

from

the

NORs

during

metaphase

and

reappear

in

anaphase

-LRB-

Chen

et

al.

Leung

et

al.

-RRB-

As

for

the

mechanism

that

governs

disassembly

of

nucleoli

in

prophase

it

may

be

assumed

that

it

is

linked

to

repression

of

rDNA

transcription

most

probably

caused

by

B-directed

phosphorylation

of

components

of

the

rDNA

transcription

machinery

-LRB-

Heix

et

al.

Sirri

et

al.

-RRB-

At

the

beginning

of

prophase

the

components

of

the

pre-rRNA

processing

machinery

do

not

remain

in

the

vicinity

of

the

rDNAs

-LRB-

Gautier

et

al.

-RRB-

but

become

partially

distributed

over

the

surface

of

all

the

chromosomes

-LRB-

reviewed

in

Hernandez-Verdun

et

al.

-RRB-

The

nucleolar

proteins

that

relocate

to

the

chromosome

periphery

are

components

of

the

DFC

and

GC

of

the

active

nucleolus

In

living

cells

nucleolar

proteins

tagged

with

GFP

are

concentrated

around

the

chromosomes

during

mitosis

and

migrate

with

the

chromosomes

-LRB-

Savino

et

al.

-RRB-

However

the

mechanisms

maintaining

interactions

of

nucleolar

processing

proteins

with

chromosomes

during

mitosis

have

not

been

characterized

The

colocalization

of

the

different

factors

involved

in

pre-rRNA

processing

suggests

that

processing

complexes

are

at

least

to

some

extent

maintained

during

mitosis

It

is

as

yet

unknown

whether

migration

of

the

nucleolar

processing

proteins

occurring

at

the

onset

of

mitosis

-LRB-

Fan

and

Penman

-RRB-

takes

place

as

a

consequence

of

the

arrest

of

pre-rRNA

synthesis

or

whether

it

is

also

regulated

Indeed

it

is

noticeable

that

-LRB-

-RRB-

during

prophase

the

components

of

the

rRNA

processing

machinery

appear

to

be

delocalized

before

total

repression

of

rDNA

transcription

occurs

and

-LRB-

-RRB-

the

most

recently

synthesized

pre-rRNAs

accumulate

as

partially

processed

pre-rRNAs

-LRB-

Dousset

et

al.

-RRB-

suggesting

that

total

repression

of

pre-rRNA

processing

could

occur

prior

to

total

repression

of

rDNA

transcription

These

observations

therefore

raise

the

possibility

that

rDNA

transcription

and

pre-rRNA

processing

are

both

repressed

during

prophase

by

distinct

mechanisms

Assembly

in

telophase

Nucleoli

assemble

at

the

exit

from

mitosis

concomitantly

with

restoration

of

rDNA

transcription

at

the

level

of

competent

NORs

-LRB-

Roussel

et

al.

-RRB-

Until

recently

it

was

admitted

that

transcriptionally

active

rDNAs

serving

as

nucleation

sites

possessed

by

themselves

the

ability

to

organize

the

nucleoli

-LRB-

Scheer

and

Weisenberger

-RRB-

Results

obtained

in

the

laboratory

showed

that

-LRB-

-RRB-

reactivation

of

rDNA

transcription

in

mitotic

cells

does

not

lead

to

the

formation

of

nucleoli

-LRB-

Sirri

et

al.

-RRB-

-LRB-

-RRB-

initiation

of

nucleolar

assembly

occurs

independently

of

rDNA

transcription

-LRB-

Dousset

et

al.

-RRB-

and

-LRB-

-RRB-

at

the

exit

from

mitosis

nucleologenesis

is

impaired

in

the

presence

of

a

CDK

inhibitor

even

if

rDNAs

are

actively

transcribed

-LRB-

Sirri

et

al.

-RRB-

Consequently

the

formation

of

functional

nucleoli

at

the

exit

from

mitosis

is

not

governed

solely

by

the

resumption

of

rDNA

transcription

Based

on

previous

studies

-LRB-

Sirri

et

al.

-RRB-

we

propose

that

the

formation

of

nucleoli

is

a

process

regulated

by

CDK

-LRB-

s

-RRB-

at

two

levels

resumption

of

rDNA

transcription

but

also

restoration

of

rRNA

processing

In

anaphase

early

and

late

processing

proteins

-LRB-

respectively

fibrillarin

and

-RRB-

are

homogeneously

distributed

around

the

chromosomes

During

telophase

and

early

along

the

translocation

pathway

between

chromosome

periphery

and

transcription

sites

processing

proteins

concentrate

in

foci

designated

prenucleolar

bodies

-LRB-

PNBs

-RRB-

first

described

in

plant

cells

-LRB-

Stevens

-RRB-

PNB

formation

is

a

general

phenomenon

occurring

during

the

recruitment

of

the

nucleolar

processing

proteins

at

exit

from

mitosis

-LRB-

Angelier

et

al.

Azum-Gélade

et

al.

Dundr

et

al.

Jiménez-Garcia

et

al.

Ochs

et

al.

Savino

et

al.

-RRB-

This

appears

to

be

a

cell

cycle

regulated

process

since

when

the

nucleolar

function

is

established

during

interphase

recruitment

of

processing

proteins

is

not

associated

with

PNB

formation

Inactivation

of

B

occurring

at

the

end

of

mitosis

induces

the

first

events

of

nucleologenesis

Strikingly

fibrillarin

concentrates

in

PNBs

and

rDNA

clusters

when

decrease

in

B

activity

overcomes

the

mitotic

repression

of

RNA

pol

I

transcription

-LRB-

Clute

and

Pines

-RRB-

while

and

other

GC

proteins

are

recruited

later

on

transcription

sites

This

late

recruitment

is

under

the

control

of

cyclin-dependent

kinases

since

CDK

inhibitors

block

this

process

-LRB-

Sirri

et

al.

-RRB-

Thus

it

seems

that

recruitment

of

the

processing

machinery

at

the

time

of

nucleolar

assembly

is

a

regulated

process

most

probably

dependent

on

cell

cycle

progression

This

provides

a

physiological

situation

to

investigate

the

formation

control

and

dynamics

of

nuclear

bodies

The

dynamics

of

the

processing

nucleolar

proteins

was

analyzed

at

the

transition

mitosis/interphase

using

rapid

time-lapse

video

microscopy

-LRB-

Fig.

-RRB-

The

first

detectable

assembly

of

proteins

in

foci

occurred

on

the

surface

of

the

chromosome

during

telophase

-LRB-

Savino

et

al.

-RRB-

followed

by

the

progressive

delivery

of

proteins

to

nucleoli

ensured

by

progressive

and

sequential

release

of

proteins

from

PNBs

-LRB-

Dundr

et

al.

-RRB-

Based

on

the

observations

of

different

fixed

cells

it

was

concluded

that

the

early

processing

proteins

are

recruited

first

on

transcription

sites

while

the

majority

of

the

late

processing

proteins

are

still

in

PNBs

-LRB-

Fomproix

et

al.

Savino

et

al.

-RRB-

This

sequence

of

events

was

confirmed

in

living

HeLa

cells

Fibrillarin

resides

briefly

in

PNBs

-LRB-

min

-RRB-

before

recruitment

to

the

nucleolus

while

is

maintained

longer

in

PNBs

-LRB-

min

-RRB-

-LRB-

Savino

et

al.

-RRB-

The

relative

dynamics

of

early

and

late

rRNA

processing

proteins

at

the

time

of

PNB

formation

was

examined

using

co-expression

of

GFP-fibrillarin

and

-LRB-

Angelier

et

al.

-RRB-

Once

near

the

poles

min

after

the

onset

of

telophase

numerous

bright

fluorescent

foci

containing

both

GFP-fibrillarin

and

appeared

almost

simultaneously

For

about

min

the

relative

amount

of

in

foci

was

five

to

six

times

higher

than

that

of

the

dispersed

proteins

whereas

the

amount

of

fibrillarin

in

the

same

foci

was

three

to

four

times

higher

than

that

of

dispersed

proteins

Subsequently

fibrillarin

was

released

while

was

still

present

in

the

foci

This

clearly

illustrates

the

presence

of

the

two

types

of

nucleolar

processing

proteins

in

the

same

PNBs

and

suggests

differential

sorting

of

these

proteins

Conversely

in

the

same

conditions

similar

dynamics

and

flows

of

and

were

observed

Thus

the

processing

proteins

passed

through

the

same

PNBs

and

were

released

simultaneously

suggesting

that

these

proteins

could

form

complexes

in

PNBs

Fig.

the

exit

from

mitosis

the

dynamics

of

is

followed

in

living

cell

In

telophase

-LRB-

min

-RRB-

the

signal

is

visible

in

small

foci

These

foci

corresponding

to

PNBs

are

clearly

visible

min

later

The

PNBs

are

distributed

in

the

nucleoplasm

and

is

progressively

recruited

in

the

incipient

nucleolus

-LRB-

min

-RRB-

Nu

nucleolus

Time-lapse

analysis

of

fluorescence

resonance

energy

transfer

-LRB-

FRET

-RRB-

was

chosen

to

determine

whether

nucleolar

processing

proteins

interact

along

the

recruitment

pathway

The

apparatus

used

to

determine

FRET

performs

tdFLIM

-LRB-

time

domain

fluorescence

lifetime

imaging

microscopy

-RRB-

by

the

time

and

space-correlated

single-photon

counting

method

-LRB-

Emiliani

et

al.

-RRB-

This

technique

directly

yields

the

picosecond

time-resolved

fluorescence

decay

for

every

pixel

by

counting

and

sampling

single

emitted

photons

Positive

FRET

between

and

in

nucleoli

indicates

that

the

distance

and

most

probably

the

interactions

between

the

proteins

can

be

evaluated

by

this

approach

-LRB-

Angelier

et

al.

-RRB-

Since

it

is

possible

to

detect

FRET

between

and

in

nucleoli

FRET

was

tracked

during

the

recruitment

of

these

proteins

into

nucleoli

from

anaphase

to

early

FRET

was

never

detected

during

anaphase

at

the

periphery

of

the

chromosomes

whereas

it

was

registered

in

of

the

PNBs

at

the

beginning

of

telophase

in

about

at

the

end

of

telophase

and

in

in

early

Thus

interaction

between

and

was

established

progressively

in

PNBs

as

the

number

of

PNBs

exhibiting

FRET

increased

Such

data

indicate

that

and

did

not

interact

until

they

were

recruited

in

PNBs

It

is

noteworthy

that

a

given

PNB

can

alternatively

present

or

not

present

FRET

Based

on

the

behavior

of

these

two

proteins

one

possibility

is

that

late

rRNA

processing

proteins

already

interact

in

PNBs

Were

this

to

be

confirmed

for

other

rRNA

processing

complexes

PNBs

could

be

proposed

as

assembly

platforms

of

processing

complexes

at

this

step

of

the

cell

cycle

It

would

be

very

interesting

to

establish

whether

this

role

can

be

extended

to

the

early

rRNA

processing

machinery

-LRB-

Angelier

et

al.

-RRB-

In

conclusion

assembly

of

the

nucleolus

requires

reactivation

of

the

rDNA

transcription

machinery

and

also

recruitment

and

reactivation

of

the

pre-rRNA

processing

machinery

Indeed

cells

exiting

from

mitosis

in

the

presence

of

a

CDK

inhibitor

exhibit

neither

relocalization

of

the

late

pre-rRNA

processing

components

from

PNBs

to

rDNA

transcription

sites

resumption

of

proper

rRNA

processing

nor

formation

of

functional

nucleoli

Nucleolus

and

cancer

The

link

between

cell

proliferation

cancer

and

nucleolar

activity

has

been

well

established

during

the

past

several

decades

-LRB-

more

than

references

-RRB-

Half

of

the

studies

related

to

the

nucleolus

and

cancer

are

dedicated

to

the

prognostic

value

of

AgNOR

staining

a

technique

revealing

the

amount

of

nucleolar

proteins

The

aim

of

this

technique

is

to

evaluate

the

proliferation

potential

of

cancer

cells

by

measuring

nucleolar

activity

nucleolin

UBF

and

subunits

of

RNA

pol

I

were

found

to

be

the

argyrophilic

proteins

responsible

for

the

silver-staining

properties

of

nucleolar

structures

-LRB-

Roussel

et

al.

Roussel

and

Hernandez-Verdun

-RRB-

In

interphase

cells

the

amount

of

major

AgNOR

proteins

and

nucleolin

is

high

in

S

and

low

in

phases

and

thus

a

higher

value

of

AgNOR

corresponds

to

actively

cycling

cells

-LRB-

Sirri

et

al.

-RRB-

Standardization

of

the

AgNOR

staining

method

permits

routine

application

of

this

technique

for

clinical

purposes

The

size

of

the

nucleolus

is

generally

enlarged

in

cancer

cells

and

this

has

been

correlated

with

cell

proliferation

A

new

field

of

research

was

recently

opened

by

the

discovery

that

several

tumor

suppressors

and

proto-oncogenes

affect

the

production

of

ribosomes

-LRB-

Ruggero

and

Pandolfi

-RRB-

rRNA

synthesis

is

enhanced

by

c-Myc

-LRB-

Arabi

et

al.

-RRB-

and

it

was

proposed

that

this

stimulation

is

a

key

pathway

driving

cell

growth

and

tumorigenesis

-LRB-

Grandori

et

al.

-RRB-

On

the

contrary

the

decrease

of

ribosome

production

induces

apoptosis

in

a

or

independent

manner

-LRB-

David-Pfeuty

et

al.

Pestov

et

al.

-RRB-

and

the

disruption

of

the

nucleolus

mediates

stabilization

-LRB-

Rubbi

and

Milner

-RRB-

The

cross

talk

between

the

pathway

and

the

nucleolus

is

at

least

in

part

mediated

by

localization

of

in

the

nucleolus

an

ubiquitin

ligase

involved

in

degradation

Nucleostemin

a

nucleolar

protein

discovered

in

stem

cells

and

in

cancer

cells

interacts

with

-LRB-

Tsai

and

McKay

-RRB-

It

was

proposed

that

nucleostemin

might

regulate

function

through

shuttling

between

the

nucleolus

and

the

nucleoplasm

The

major

nucleolar

protein

is

directly

implicated

in

cancer

pathogenesis

as

demonstrated

by

mutation

of

the

gene

in

a

number

of

hematological

disorders

-LRB-

Grisendi

et

al.

-RRB-

Importantly

in

acute

promyelocytic

leukemia

the

fusion

protein

NPM/RARα

localizes

in

the

nucleolus

indicating

a

role

of

this

nucleolar

protein

in

this

disease

-LRB-

Rego

et

al.

-RRB-

Nucleolus

and

virus

Within

the

last

few

years

increasing

evidence

has

revealed

that

viruses

require

the

nucleus

and

in

particular

the

nucleolus

to

target

proteins

indispensable

for

their

replication

An

increasing

number

of

key

proteins

from

both

DNA

and

RNA-containing

viruses

are

localized

in

the

nucleolus

viruses

of

the

family

Herpesviridae

Adenoviridae

Hepadnaviridae

Retroviridae

Rhabdoviridae

Orthomyxoviridae

Potyviridae

Coronaviridae

and

Flaviviridae

encode

such

proteins

Viruses

have

developed

different

strategies

to

facilitate

targeting

of

their

proteins

to

the

nucleolus

-LRB-

-RRB-

it

was

reported

that

the

sequences

of

certain

viral

proteins

harbor

NoLS

and

NES

-LRB-

Harris

and

Hope

Hiscox

Kann

et

al.

-RRB-

Recently

-LRB-

Reed

et

al.

-RRB-

it

was

demonstrated

by

mutagenesis

that

the

nucleocapsid

-LRB-

N

-RRB-

protein

of

infectious

bronchitis

virus

-LRB-

IBV

-RRB-

presents

an

amino

acid-long

motif

that

functions

as

NoLS

and

is

necessary

and

sufficient

for

nucleolar

retention

of

the

N

protein

and

colocalization

with

nucleolin

and

fibrillarin

the

NoLS

is

required

for

interaction

with

cell

factors

-LRB-

-RRB-

Other

viral

proteins

present

sequences

rich

in

arginine

lysine

-LRB-

Ghorbel

et

al.

Reed

et

al.

-RRB-

known

to

be

nucleolar

retention

signals

generally

these

sequences

overlap

the

NLS

-LRB-

-RRB-

Some

viral

proteins

that

target

the

nucleolus

present

motifs

with

affinity

for

double-stranded

RNA

-LRB-

dsRNA

-RRB-

for

RNA

binding

or

for

DNA

binding

-LRB-

Melen

et

al.

-RRB-

-LRB-

-RRB-

Other

studies

showed

that

nucleolar

localization

of

viral

proteins

is

cell

cycle-dependent

-LRB-

Cawood

et

al.

-RRB-

using

synchronization

studies

coupled

to

live

cell

confocal

microscopy

the

authors

demonstrated

that

the

concentration

of

N

protein

in

the

nucleolus

was

higher

in

the

phase

than

in

other

phases

and

that

in

this

phase

the

protein

was

more

mobile

in

the

nucleoplasm

In

all

the

cases

examined

the

viral

proteins

depend

on

cell

factors

to

successfully

shuttle

between

the

nucleolus

and

the

cytoplasm

Why

must

viral

proteins

target

to

the

nucleolus

The

answer

to

this

question

is

not

clear

however

different

authors

had

reported

that

such

viral

proteins

are

involved

both

in

replication

of

the

viral

genome

and

in

transcriptional

and

post-transcriptional

regulation

of

viral

genome

expression

-LRB-

Dang

and

Lee

Pyper

et

al.

-RRB-

For

example

some

plants

viruses

are

known

to

encode

a

protein

designated

movement

protein

responsible

for

long-distance

movement

of

the

viral

RNA

through

the

phloem

-LRB-

Ryabov

et

al.

-RRB-

Movement

strictly

depends

on

the

interaction

of

the

viral

movement

protein

with

the

nucleolus

and

the

Cajal

bodies

which

contain

snRNPs

and

snoRNPs

-LRB-

Kim

et

al.

b

-RRB-

The

open

reading

frame

-LRB-

ORF

-RRB-

of

Groundnut

rosette

virus

is

one

such

protein

it

is

first

localized

in

Cajal

bodies

and

forms

Cajal

body-like

structures

it

is

then

localized

in

the

nucleolus

when

the

Cajal

body-like

structures

fuse

with

the

nucleolus

and

finally

it

exits

to

the

cytoplasm

-LRB-

Kim

et

al.

-RRB-

Another

study

showed

that

this

shuttling

is

indispensable

to

form

the

RNPs

essential

for

systemic

virus

infection

-LRB-

Kim

et

al.

-RRB-

In

this

process

the

interaction

of

the

viral

with

fibrillarin

is

absolutely

required

Interestingly

silencing

of

the

fibrillarin

gene

blocks

long-distance

movement

of

the

virus

but

does

not

affect

virus

replication

and

movement

via

plasmodesmata

Because

the

mobility

of

nucleolar

components

depends

on

the

interactions

and

functions

of

the

components

-LRB-

Olson

and

Dundr

-RRB-

we

suggest

that

targeting

of

viral

proteins

to

the

nucleolus

could

help

viral

protein

traffic

and

diffusion

of

viral

infection

The

activity

of

the

human

immunodeficiency

virus

-LRB-

HIV

-RRB-

Rev

protein

is

essential

for

virus

replication

Its

subcellular

localization

is

nucleolar

but

it

has

the

ability

to

shuttle

continuously

between

the

nucleus

and

the

cytoplasm

-LRB-

Felber

et

al.

Kalland

et

al.

-RRB-

Rev

possesses

both

an

NES

and

an

NLS

the

NLS

is

associated

with

importin-β

as

well

as

with

-LRB-

Fankhauser

et

al.

Henderson

and

Percipalle

-RRB-

Rev-GFP

movement

in

the

nucleolus

is

very

slow

implying

that

it

is

attached

to

affinity

binding

sites

in

this

subcellular

compartment

-LRB-

Daelemans

et

al.

-RRB-

In

addition

the

transport

of

Rev

from

the

nucleolus

to

the

cytoplasm

can

be

affected

negatively

by

a

cellular

protein

that

colocalizes

with

Rev

in

the

nucleolus

-LRB-

Urcuqui-Inchima

et

al.

-RRB-

-LRB-

Fig.

-RRB-

This

indicates

that

the

transport

of

HIV

transcripts

by

Rev

to

the

cytoplasm

is

a

regulated

process

Because

Rev

is

concentrated

in

the

nucleolus

it

was

suggested

that

the

passage

of

Rev

to

the

nucleolus

is

an

indispensable

step

for

Rev

function

and

hence

for

replication

Indeed

based

on

RNA

trafficking

through

the

nucleolus

this

organelle

is

an

essential

participant

of

RNA

export

-LRB-

Michienzi

et

al.

-RRB-

Fig.

Rev-GFP

and

fusions

were

expressed

in

HeLa

cells

Both

proteins

colocalize

in

nucleoli

as

seen

by

the

merge

The

nucleus

is

visualized

by

Dapi

staining

Bars

μm

As

discussed

above

for

Rev

it

has

been

shown

that

the

Herpes

virus

saimiri

protein

is

required

for

nuclear

export

of

viral

intronless

mRNAs

-LRB-

Boyne

and

Whitehouse

-RRB-

In

addition

the

expression

of

induces

nuclear

trafficking

which

is

essential

for

nuclear

export

of

such

RNAs

the

human

transcription/export

protein

involved

in

mRNA

export

is

redistributed

to

the

nucleolus

in

the

presence

of

the

protein

Based

on

these

findings

the

authors

concluded

that

the

nucleolus

is

required

for

nuclear

export

of

the

viral

mRNAs

What

are

the

consequences

for

the

cells

of

the

passage

of

viral

proteins

via

the

nucleolus

It

is

known

that

all

viruses

whether

with

DNA

or

RNA

genomes

interfere

with

the

cell

cycle

affecting

host-cell

functions

and

increasing

the

efficiency

of

virus

replication

The

data

obtained

suggest

that

targeting

of

virus

proteins

to

the

nucleolus

not

only

facilitates

virus

replication

but

may

also

be

required

for

pathogenic

processes

Recent

studies

following

infection

by

IBV

revealed

a

change

in

the

morphology

and

protein

content

of

the

nucleolus

-LRB-

Dove

et

al.

-RRB-

This

included

an

enlarged

FC

and

an

increase

in

protein

content

interestingly

the

tumor

suppressor

protein

normally

localized

in

the

nucleus

in

virus

infected

cells

was

redistributed

mainly

in

the

cytoplasm

The

Hepatitis

B

virus

-LRB-

HBV

-RRB-

core

antigen

-LRB-

HBcAg

-RRB-

is

responsible

for

export

of

the

virus

with

a

mature

genome

-LRB-

Yuan

et

al.

b

-RRB-

Indeed

a

change

from

isoleucine

to

leucine

in

position

-LRB-

-RRB-

of

HBcAg

causes

the

cell

to

release

virus

particles

with

immature

genomes

HBcAg

with

a

mutation

in

position

-LRB-

or

-RRB-

has

been

detected

in

the

nucleolus

colocalizing

with

nucleolin

and

and

this

colocalization

was

often

related

with

binucleated

cells

or

apoptosis

-LRB-

Ning

and

Shih

-RRB-

suggesting

that

the

localization

of

HBcAg

in

the

nucleolus

could

perturb

cytokinesis

The

authors

propose

that

this

event

may

be

associated

with

liver

pathogenesis

Some

factors

expressed

by

west

nile

virus

-LRB-

WNV

-RRB-

such

as

and

and

the

WNV

capsid

-LRB-

WNVCp

-RRB-

participate

in

WNV-mediated

apoptosis

-LRB-

Oh

et

al.

Ramanathan

et

al.

-RRB-

It

is

well

known

that

is

activated

in

response

to

oncogenic

or

DNA

damaging

stresses

inducing

cell

cycle

arrest

and

apoptosis

-LRB-

Harris

and

Levine

-RRB-

In

normal

conditions

targets

and

blocks

abnormal

accumulation

of

by

ubiquitinylation

followed

by

proteasome-dependent

degradation

of

-LRB-

Haupt

et

al.

Kubbutat

et

al.

-RRB-

Recently

it

was

demonstrated

that

WNVCp

could

bind

to

and

sequester

in

the

nucleolus

blocking

complex

formation

-LRB-

Yang

et

al.

-RRB-

This

phenomenon

causes

stabilization

of

and

Bax

activation

and

thereafter

apoptosis

In

addition

the

authors

show

that

WNVCp

is

able

to

induce

apoptosis-dependent

processes

suggesting

that

the

viral

protein

mediates

apoptosis

through

mechanisms

by

retention

of

in

the

nucleolus

Remarks

and

perspectives

The

conclusions

are

based

on

the

perspectives

and

the

tendency

that

can

be

anticipated

from

the

present

research

in

the

field

of

the

nucleolus

We

propose

that

in

the

future

a

better

understanding

of

the

complexity

and

variability

of

ribosome

biogenesis

will

need

to

be

established

For

example

the

difference

between

the

information

available

in

yeast

and

mammalian

cells

is

of

major

importance

The

different

steps

of

ribosome

biogenesis

and

protein

complexes

are

well

characterized

in

yeast

due

to

easy

access

of

mutants

Similarly

Miller

chromatin

spreading

for

electron

microscopy

in

yeast

strains

carrying

mutations

reveals

the

coupling

of

RNA

pol

I

transcription

with

rRNA

processing

-LRB-

Schneider

et

al.

-RRB-

Additionally

the

compaction

into

SSU

processomes

of

ribosomal

RNA

before

cleavage

was

observed

on

Miller

spreads

-LRB-

Osheim

et

al.

-RRB-

There

is

presently

no

comparable

information

for

the

mammalian

genes

Yet

the

tendency

is

to

generalize

and

suppose

that

the

information

is

similar

in

the

two

models

In

the

future

differences

will

most

probably

be

revealed

as

well

as

the

complexity

of

the

regulation

in

differentiated

cells

Along

this

line

it

was

demonstrated

that

basonuclin

a

cell-type-specific

rDNA

regulator

transcribes

only

one

subset

of

rDNAs

of

a

cell

-LRB-

Zhang

et

al.

-RRB-

In

such

a

differentiated

cell

it

remains

to

be

established

how

the

subset

of

rDNA

repeats

is

selected

The

nucleolus

was

proposed

to

be

a

domain

of

the

sequestration

of

molecules

that

normally

operate

outside

this

organelle

mainly

in

the

nucleoplasm

Sequestration

in

the

yeast

nucleolus

of

the

phosphatase

and

its

release

into

the

cytoplasm

at

anaphase

was

demonstrated

to

be

a

key

event

in

cell

cycle

progression

-LRB-

for

a

review

see

Cockell

and

Gasser

Guarente

Visintin

and

Amon

-RRB-

However

it

is

important

to

recall

that

there

is

no

nucleolus

during

mitosis

in

mammalian

cells

In

mammalian

interphase

cells

the

nucleolus

is

a

domain

of

retention

of

molecules

related

to

cell

cycle

life

span

and

apoptosis

and

is

in

particular

an

actor

of

the

pathway

Recently

nucleolar

retention

of

the

transcription

factor

was

observed

in

trophoblast

stem

cells

-LRB-

Martindill

et

al.

-RRB-

Phosphorylation

of

induced

nucleolar

to

nucleoplasm

translocation

of

and

commitment

of

stem

cells

to

differentiate

into

giant

cells

translocation

to

the

nucleoplasm

might

regulate

a

crucial

step

of

stem

cell

differentiation

into

polyploid

giant

cells

but

the

targets

of

in

the

nucleoplasm

are

still

undefined

The

nucleolus

is

generally

surrounded

by

highly

condensed

chromatin

first

described

in

rat

hepatocytes

and

presently

known

as

heterochromatin

By

following

the

movements

of

chromosome

sequences

introduced

in

different

sites

in

chromosomes

of

living

cells

it

was

demonstrated

that

loci

at

nucleoli

periphery

and

nuclear

periphery

are

less

mobile

than

in

other

sites

Disruption

of

the

nucleoli

by

a

inhibitor

increases

the

mobility

of

the

perinucleolar

loci

It

was

proposed

that

the

nucleolus

and

nuclear

periphery

could

maintain

the

three-dimensional

organization

of

chromatin

in

the

nucleus

-LRB-

Chubb

et

al.

-RRB-

Recently

the

perinucleolar

ring

of

chromatin

was

brought

to

the

fore

when

its

role

in

the

maintenance

of

inactive

X

-LRB-

Xi

-RRB-

was

demonstrated

-LRB-

Zhang

et

al.

-RRB-

During

middle

and

late

S

phase

Xi

contacts

the

nucleolar

periphery

when

it

is

replicated

during

the

cell

cycle

It

was

discovered

that

the

perinucleolar

chromatin

is

enriched

in

the

catalytic

subunit

of

a

remodeling

complex

required

for

replication

of

heterochromatin

These

observations

demonstrate

the

role

of

the

perinucleolar

compartment

in

maintaining

the

epigenetic

state

of

Xi

-LRB-

Zhang

et

al.

-RRB-

The

presence

of

inactive

rDNA

repeats

in

perinucleolar

heterochromatin

is

known

in

many

plant

cells

and

in

Drosophila

It

was

recently

demonstrated

that

disruption

in

Drosophila

of

histone

methylation

a

marker

of

heterochromatin

induced

nucleolar

disorganization

and

decondensation

and

disorganization

of

rDNA

repeats

-LRB-

Peng

and

Karpen

-RRB-

The

authors

suggest

that

condensation

of

a

part

of

the

rDNA

copies

into

heterochromatin

could

be

a

general

strategy

against

recombination

of

these

highly

repeated

genes

For

long

interest

concerning

the

nucleolus

was

to

establish

how

efficient

ribosome

biogenesis

occurs

and

the

link

of

this

function

with

the

cell

cycle

More

recently

the

effect

of

the

disruption

of

ribosome

biogenesis

appeared

very

important

when

it

was

proposed

that

the

nucleolus

is

a

sensor

of

stress

-LRB-

Rubbi

and

Milner

-RRB-

Indeed

disruption

of

ribosome

biogenesis

releases

ribosomal

proteins

from

the

nucleolus

that

bind

to

and

inhibit

degradation

-LRB-

Lindstrom

et

al.

-RRB-

A

connection

between

ribosomal

stress

and

cell

cycle

arrest

is

now

proposed

-LRB-

Gilkes

et

al.

-RRB-

Considering

the

diversity

of

the

recent

information

gathered

on

the

nucleolus

it

is

clear

that

this

is

a

very

dynamic

and

rapidly

progressing

research

area

The

most

promising

aspect

is

the

contribution

of

new

models

-LRB-

pseudo-NORs

Prieto

and

McStay

-RRB-

new

species

-LRB-

not

only

yeast

-RRB-

new

approaches

-LRB-

Miller

spreads

using

mutants

proteomics

-RRB-

and

new

questions

-LRB-

for

instance

the

role

of

siRNAs

or

antisens

RNAs

in

the

activity

of

the

nucleolus

-RRB-

nucleolus assembly dynamics pnb virus cancer