biocos_febsletterpaper_jan26_2013

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Characterization of nucleolin K88 acetylation defines a new poolof nucleolin colocalizing with pre-mRNA splicing factors

Sadhan Das a, Rong Cong a, Jayasha Shandilya b, Parijat Senapati b, Benoit Moindrot a, Karine Monier a,Hlne Delage a, Fabien Mongelard a, Sanjeev Kumar c, Tapas K. Kundu b, Philippe Bouvet a,

a Universit de Lyon, Ecole Normale Suprieure de Lyon, CNRS USR 3010, Laboratoire Joliot-Curie, Lyon, FrancebJawaharlal Nehru Centre for Advanced Scientific Research, Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Bangalore, Indiac BioCOS Life Sciences Private Limited, Biotech Park, Electronics City, Phase-1, Bangalore, India

a r t i c l e i n f o

Article history:

Received 3 September 2012

Revised 17 December 2012

Accepted 14 January 2013

Available online xxxx

Edited by Ulrike Kutay

Keywords:

Nucleolin

Post-translational modification

Acetylation

Interchromatin granule cluster

SC35

Splicing factor

a b s t r a c t

Nucleolin is a multifunctional protein that carries several post-translational modifications. We char-

acterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using

this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but

instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acet-

ylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated

nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation

defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA mat-

uration and transcription by RNA polymerase II.

Structured summary of protein interactions:

SC35 physically interacts with Nucleolin by anti bait coimmunoprecipitation (View interaction)

Nucleolin and SC35 colocalize by fluorescence microscopy (View interaction)

2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Nucleolin was first identified as one of the major nucleolar

phosphoproteins [1]. Because of its predominant nucleolar locali-

zation, the different functions of nucleolin in ribosome biogenesis

have been extensively studied [2]. In particular, nucleolin is in-

volved in the first processing step of pre-rRNA maturation

in vitro [3] and it interacts with numerous ribosomal proteins

[4]. NucleolinrRNA interaction studies suggest that nucleolin

could be involved in the co-transcriptional folding of pre-rRNA

which is necessary for the correct maturation of pre-rRNA [5]. In-deed, in nucleolin knockout DT40 cells [6], the processing of 45S

pre-rRNA is moderately affected. However, the most prominent ef-

fect of nucleolin knockout or silencing, is on pre-rRNA accumula-

tion suggesting that nucleolin could be involved in some aspects

of pre-rRNA transcription elongation [610]. Nucleolin has also

been involved in DNA repair, mRNAs metabolism, internalization

of growth factors and viral ligands and virus replication [5].

These multiple functions can be achieved via numerous pro-

teinprotein interactions, and probably also thanks to the numer-

ous post-translational modifications (PTM) whose functions are

still largely unexplored.

The best characterized PTM of nucleolin is undoubtedly the

phosphorylation of the N-terminal domain [11,12], which has been

involved in the regulation of transcription [13,14] and nucleic acid

interaction [15]. The C-terminal domain of nucleolin is the site of

NG,NG-dimethylarginine [16] that can modulate the interaction of

nucleolin with nucleic acids [17]. Nucleolin is also glycosylated

in human U397 cells [18].Acetylation is another PTM that has been extensively studied.

p300/CBP, the GNAT family of histone acetytransferase (HAT) acet-

ylates a large number of non-histone proteins [19]. Acetylome

analysis by mass spectrometry identified acetylated residues on

nucleolin [20].

In this study, we identified several acetylated lysine in the

N-terminal domain of nucleolin and we developed an antibody

specific to nucleolin acetylated lysine 88 (NCL-K88ac). Using this

antibody, we demonstrated that a fraction of nucleolin could be

acetylated in vivo. Interestingly, we found that NCL-K88ac is not

localized within the nucleolus, but rather in nucleoplasmic

speckles that colocalized with splicing factors SC35. This speckle

0014-5793/$36.00 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.febslet.2013.01.035

Corresponding author. Address: Ecole Normale Suprieure de Lyon, Laboratoire

Joliot-Curie, CNRS USR 3010, 46 Alle dItalie, 69007 Lyon, France. Fax: +33

472728016.

E-mail address: [email protected] (P. Bouvet).

FEBS Letters xxx (2013) xxxxxx

j o u r n a l h o m e p a g e : w w w . F E B S L e t t e r s . o r g

Please cite this article in press as: Das, S., et al. Characterization of nucleolin K88 acetylation defines a new pool of nucleolin colocalizing with pre-mRNA

splicing factors. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.01.035
http://dx.doi.org/10.1016/j.febslet.2013.01.035mailto:[email protected]://dx.doi.org/10.1016/j.febslet.2013.01.035http://www.febsletters.org/http://dx.doi.org/10.1016/j.febslet.2013.01.035http://dx.doi.org/10.1016/j.febslet.2013.01.035http://www.febsletters.org/http://dx.doi.org/10.1016/j.febslet.2013.01.035mailto:[email protected]://dx.doi.org/10.1016/j.febslet.2013.01.035


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localization was found in HeLa cells and in stimulated Peripheral

Blood Mononuclear Cells (PBMC).

2. Materials and methods

2.1. In vitro acetylation assay

In vitro acetylation assays were performed using 2 lg of pro-teins, incubated at 30 C for 30 min in a 30ll acetylation buffer(50 mM TrisHCl (pH 8.0), 10% (vol/vol) glycerol, 1 mM dithio-

threitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA (pH

8.0), 10 mM sodium butyrate) and 0.5 ll of 3.3 Ci/mmol [3H]acet-yl-CoA and analyzed as described in the legend of Supplementary

Fig. 1. For the mass acetylation reaction, 2 lg of baculovirus-ex-pressed nucleolin and 100 ng full-length p300 were incubated in

the presence of 50lM acetyl-CoA at 37 C for 1.5 h. To achieve effi-cient acetylation, p300 and acetyl-CoA were added at every 30-min

interval.

2.2. Generation of polyclonal antibodies specific for acetylated

nucleolin and characterization of the in vivo status of nucleolin

acetylation

Based on the identified acetylation sites by mass spectrometry,

four different peptides having acetylated lysine residues (AcK)

were designed (see sequences in Supplementary Fig. 2). Each acet-

ylated peptide was used for immunization of a rabbit (Covalab,

Lyon). The specificity of the four resulting serums was first checked

by an ELISA with the acetylated and non-acetylated peptides. The

serum obtained from the immunization with AcNcl1 peptide

showed the highest specificity, and was chosen to perform the

experiments described in this manuscript. For some experiments

(as indicated in the text) we used the purified IgG of AcNcl1 that

were purified using Protein A Agarose beads (Thermo Scientific).

Immunoprecipitation experiments using nucleolin antibodies were

done as described previously [10].

2.3. Cell culture, siRNA transfection and immunofluorescence studies

HeLa cells were cultured in Dulbeccos modified Eagle medium

(DMEM, Gibco) supplemented with 10% foetal bovine serum (FBS)

(Gibco) at 37 C in 5% CO2 incubator. HeLa cells (6 105) were

transfected twice with siRNA Ncl2 and Ncl4 (Eurogentec) using

Lipofectamine 2000 (Invitrogen) as described previously [8]. As a

siRNA control we used Stealth high GC siRNA (Invitrogen). Proteins

were harvested at 96 h after the first transfection, and analysed by

Western blot.

2.4. Antibodies

The following antibodies were used: A rabbit polyclonal anti-body againsthumannucleolin no 5567developed by our laboratory

and used previously [21], a rabbit polyclonal antibody against hu-

man acetylated nucleolin (AcNcl1) (Covalab, Lyon; this study),

anti-acetyl-Histone H3 (Lys14) (17-305,Upstate) anti-SC35

(ab11826, Abcam), anti-Y12 (ab3138, Abcam), anti-Coilin (IH10)

(ab87913, Abcam), anti-acetyl lysine (ab21623, Abcam), and mouse

monoclonal anti-nucleolin antibody (KAM-CP100, Stressgen).

3. Results

3.1. Nucleolin is acetylated both in vivo and in vitro

With the aim to determine if nucleolin could be acetylatedin vivo, we prepared a nuclear S2 extract which is the first step

of nucleolin purification from HeLa cells [21]. (Fig. 1A, B). Western

blot on this extract with global anti-acetyl Lysine antibody

(Ab21623, Abcam) clearly detects a faint band corresponding to

nucleolin size (Fig. 1C) suggesting that a fraction of nucleolin pro-

tein could be acetylated in vivo. To confirm this data, and identify

the lysine acetyltransferase(s) (KAT) that are able to post-transla-

tionaly modify nucleolin, we carried out an in vitro acetylation as-

say using different KATs and [3H]acetyl-CoA. The normalized KATs

(Supplementary Fig. 1) were tested for their ability to acetylate

nucleolin in vitro (Fig. 1D). Interestingly, it was found that only

p300 and PCAF were able to acetylate nucleolin in vitro ( Fig. 1D,

lanes 4 and 6).

3.2. Identification of nucleolin acetylation sites

We set out to identify the acetylation sites by mass-spectrome-

try. Direct analysis of the purified nucleolin or immunoprecipita-

tions with global anti-acetyl lysine antibodies were not

successful, probably because only a small fraction of nucleolin is

acetylated (see later) and because of the inefficiency of IP with

the used global-acetyl lysine antibodies. Therefore, we tried the

identification of nucleolin acetylation sites after an in vitro acety-

lation reaction using cold Acetyl CoA (see Supplementary experi-

mental procedures). Three independent experiments were

performed and several acetylated lysine sites were identified

(Fig. 2B). Interestingly, all acetylation sites were found in the first

150 amino-acids of the N-terminal domain of nucleolin.

To further characterize nucleolin acetylation, we raised acetyla-

tion-specific antibodies against acetylated peptides. Four peptides

covering 8 of these acetylation sites were designed (Supplemen-

tary Fig. 2A) and used for rabbit immunization. The anti-AcNcl1

antibody (raised against the AcNcl1 peptide) was further studied

in this manuscript. The specificity of AcNcl1 serum was then char-

acterized by ELISA with the AcNcl1 modified and unmodified pep-

tide used for immunization (Supplementary Fig. 2B). These data

clearly indicate that the AcNcl1 serum is specifically directed

against the acetyl modification of the peptide. In addition, crossreactivity of AcNcl1 serum with AcNcl2, AcNcl3 and AcNcl4 modi-

fied peptides was also studied (Supplementary Fig. 2C) and the

reactivity toward acetylated histones was determined by Western

blot (Supplementary Fig. 2D). These experiments show that AcNcl1

serum present a good specificity only for the AcNcl1 peptide. Since

AcNcl1 peptide contains 2 acetylated Lysines (K79 and K88), ELISA

tests were performed with two new peptides carrying only one of

the acetylated Lysine (Supplementary Fig. 3). Remarkably, only the

peptide AcNcl1_Pep1 carrying acetylated K88 reacted efficiently

(and as AcNcl1 peptide) with the AcNcl1 serum, indicating that this

serum contains antibodies specific to this nucleolin K88 acetylated

lysine (NCL-K88ac).

Then, the specificity of this AcNcl1 antibody was characterized

by Western blot analysis using full-length nucleolin purified frombaculovirus that was subjected or not to an in vitro acetylation

reaction with p300 (Fig. 2C). AcNcl1 antibody reacts only very

weakly with baculovirus purified nucleolin (lane 1) whereas a

strong signal is detected after the acetylation reaction (lane 2). In

addition, with a HeLa whole cell extract, AcNcl1 serum detects only

one weak protein bands at the size of nucleolin (Fig. 2D). These

experiments suggest that this AcNcl1 serum is specific of nucleolin

acetylation and that the level of nucleolin acetylation is probably

low in the cell.

To further demonstrate that AcNcl1 antibody is specific to

nucleolin, we performed a series of immuno-precipitation (IP)

experiments from HeLa cells with AcNcl1 serum and with another

previously well characterized polyclonal nucleolin antibody [21]

(Fig. 2E). After an IP with AcNcl1 serum, nucleolin Ab gives a strongsignal (lane 5, second row), and inversely after IP with nucleolin

2 S. Das et al. / FEBS Letters xxx (2013) xxxxxx


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Ab, AcNcl1 is able to recognize the nucleolin immunoprecipated

protein (lane 4, first row). As previously shown, AcNcl1 serum

gives only a weak signal with total input proteins, suggesting

again that the level of NCL-K88ac is low in cells. Altogether, these

experiments show that this AcNcl1 serum is specific to nucleolin

acetylation and that nucleolin is indeed acetylated in vivo.

3.3. NCL-K88ac localizes in the nucleoplasm and is colocalized with

different nuclear markers

We used our AcNcl1 Ab to determine the cellular localization of

NCL-K88ac by immunofluorescence (Fig. 3). Interestingly, AcNcl1

Ab did not label the nucleoli structures as typical nucleolin antibod-

ies do, but instead detected nucleoplasmic speckles (Fig. 3A). Thenucleolin polyclonal antibody which strongly labels the nucleoli

also gives some faint signal in the nucleoplasm that colocalize with

the signal of AcNcl1 Ab (Fig. 3A) which confirm the Western blot

analysis (Fig. 2E) showing that the polyclonal Ab recognize also

the pool of acetylated nucleolin. The signal specificity was demon-

strated by treating the cells with nucleolin siRNA (Supplementary

Fig. 4). Inhibition of deacetylases with a sodium butyrate (NaBu)

treatment (Fig. 3BE) leads to a twofold increase of the signal ob-

served by Western blot (Fig. 3B and C) and immunofluorescence

(Fig. 3D and E) with the AcNcl1Ab whereasthe signal obtained with

the nucleolin polyclonal Ab is not changed (Fig. 3B). Since global

nucleolin Ab detects mainly nucleolin in thenucleoli and associated

with the coding region of rDNA chromatin using ChIP-seq [10] we

performed a ChIP-seq analysis of NCL-K88ac interaction with rDNA(Supplementary Fig. 5). In contrast with the interactionof nucleolin

with rDNA, NCL-K88ac does not seem significantly bound to rDNA

chromatin. Altogether, these data confirm that AcNcl1 Ab is specific

of nucleolin acetylation and show that NCL-K88ac is excluded from

the nucleolus and predominantly localized in the nucleoplasm.

To identify the nucleoplasmic structures labeled with AcNcl1

Ab, we studied the distribution of the AcNcl1 antibody and differ-

ent nuclear markers (Fig. 4) that are also known to give a speckle-

like distribution such as the splicing factor SC35 and the snRNP

marker Y12. The amount of colocalization was quantified by NIH

ImageJ software and is depicted as Pearsons coefficient (Fig. 4, bot-

tom right insert of each panel, and 4D) where a number near +1

suggests perfect correlation between 2 biomolecules, and a num-

ber near 0 indicates no correlation. Strikingly, the signal obtained

with AcNcl1 Ab is almost completely co-localized with SC35(Fig. 4A) and partially with Y-12 (Fig. 4B). In contrast, AcNcl1 Ab

did not co-localize at all with coilin (Fig. 4C). In agreement with

the co-localization of NCL-K88ac and SC35, immunoprecipitation

with an anti-SC35 antibody is able to pull down NCL-K88ac as de-

tected by AcNcl1 Ab (Supplementary Fig. 6), suggesting that NCL-

K88ac and SC35 are indeed present in the same cellular structures.

3.4. Acetylation of nucleolin in different cell lines

The level of NCL-K88ac was checked in different cells (Fig. 5A).

Acetylated nucleolin as detected by AcNcl1 seems absent fromrest-

ingPBMC(lane1) butis presentin stimulatedcells (lane 2). Interest-

ingly, the level of expression of NCL-K88ac follows the level of

expression of nucleolin in the different cell lines as the ratio of thesignal AcNcl1/Ncl is very similar in all tested cell lines (Fig. 5B). Like

A

S2Ex

trac

t

Marker

Nucleolin

170

100

130

KDa

Coomassie staining

B

WB:Nucleolin

S2Ex

trac

t C

S2Ex

trac

t

WB: acetylated-Lysine

70

55

40

30

Coomassie

Autoradio gram PCAF

autoacetylation

Nucleolin

Nucleolin

Nucleolin

[3H]-Acetyl CoA

PAT

-+

p300

+-

p300

+

-

-

++

p300

++

CBP

++

PCAF

++

Gcn5

++

Tip60

++

Moz

1 2 3 4 5 6 7 8 9

*

*

D

Fig. 1. Acetylation of nuclear S2 extract and nucleolin proteins. (A) Analysis of nuclear S2 proteins (15 lg) separated on a 10% SDSPAGE and stained with Coomassie blue.Molecular weight markers (kDa) are indicated on the left of the figure. (B) Endogenous nucleolin from HeLa nuclear S2 extract revealed by Western blot with anti-nucleolin

antibody 5567. (C) Acetylation of proteins of the HeLa nuclear S2 extract revealed by Western blot with anti-pan acetylated lysine antibody (Abcam 21623). (D) Nucleolin is

acetylated in vitro by p300 and PCAF. In vitro acetylation assay was carried out with nucleolin as substrate in the absence (lane 3) or presence of p300 (lanes 1, 2 and 4). The

acetylation reactions were also performed with differentPATsas indicatedon the figure.Lanes 1, 4, 5, 6, 7, 8 and9 show theacetylationreactionin the presence of [3H]acetyl-

CoA and lanes 2 and 3 in the absence of [3H]acetyl-CoA. Reaction products were resolved on a 10% SDSPAGE. The gel was stained with Coomassie blue to demonstrate that

equivalent substrate was used in each reaction (lower panels), and subsequently [3H]acetate reaction products were visualized by autoradiography of the same gel (upper

panels). Note the autoacetylation of PCAF (lane 6) indicated by an asterisk.

S. Das et al. / FEBS Letters xxx (2013) xxxxxx 3


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inHeLa cells (Fig.3A), acetylated nucleolin detected by AcNcl1 Ab is

perfectly co-localized with the splicing factor SC35 in stimulated

PBMC (Fig. 5C).

4. Discussion

In this report, we show that nucleolin is acetylated in vivo.

We identified several acetylated lysines, which are present

exclusively within the first 150 N-terminal residues of nucleolin.

Other acetylated residues may however exist as the purpose of

this work was not to have an exhaustive analysis of nucleolin

PTM. Also, it is not known if all these modifications are present

simultaneously in the same molecules. NCL-K88ac represent a

distinct pool of protein with a distinct cellular localization:

NCL-K88ac is apparently absent from the nucleolar structures,

but is present in speckle structures in the nucleoplasm (Figs. 3

and 4).

The absence of NCL-K88ac from the nucleoli suggests that it is

not involved in ribosome biogenesis and, in particular, in the reg-

ulation of transcription by RNAPI. ChIP-Seq with a polyclonal anti-

body show that nucleolin is associated with the coding region of

rDNA, similar to the distribution of UBF and RNAPI subunit

RPA116 [10,22]. However, ChIP-Seq with AcNcl1 does not detect

any significant binding of NCL-K88ac on rDNA chromatin

(Supplementary Fig. 5). This finding is in agreement with the

MVKLAKAGKNQGDP KKMAPPPKEVEEDSEDEEMSEDEEDDSSGEEVVIPQ

KKGKKAAATSAKKV VVSP TKKVAVATPAKKAAVTPGKKAAATPAKKTVTP

AKAVTTPGKK GATPGKALVATPGKKGAAIPAKGAKNGKNAKKEDSDEEED

DDSEEDEEDDEDEDEDEDEIEPAAMKAAAAAAPASEDEDDEDDEDDEDDD

1

51

101

50

100

150

151 200

B

Nuclear Localization Signal

Acidic st retches

Basic stretches RNA binding domain

GAR domain

N-ter Central domain C-ter

Nucleolin

aa: 1 100 200 300 400 500 600 700

A

C

WB: AcNcl1

WB: Nucleolin

1 2 3 4 5

E

AcNc

l1

Inpu

t

No

ab

IgG Nuc

leo

lin

IP:

D WCE

130

100

KDa

WB: AcNcl1

Bac-Nuc

leo

lin

Ac-Bac-Nucleo

lin

WB: Nucleolin

WB:AcNcl1

1 2

Fig. 2. Identificationof nucleolin acetylation sites by mass spectrometry and characterization of anti-ac-nucleolin (AcNcl1) antibody. (A) Schematic diagram of the full-length

nucleolinstructure, positions of amino acids1700 weremarked. (B) Protein sequence of the N-terminal domain of nucleolinshowing the acetylation sites (K9, K15, K63, K70,

K79, K80, K87, K88, K102, K109, K110, K116, K124, K125, and K135) identified by mass spectrometry. The lysines that are acetylated are marked in red while non-acetylated

lysines are left unmarked. (C) Western blot using the AcNcl1 serum (Crude serum) with the baculovirus-expressed nucleolin before (lane 1) or after (lane 2) in vitro

acetylation with p300. (D) HeLa whole cell extract (WCE) was subjected to Western blot analysis with AcNcl1 antibody. (E) Immunoprecipitation (IP) assay was performed

with HeLa cells with anti-nucleolin 5567 and AcNcl1 antibody. Following IP, Western blot was done with either AcNcl1 or anti-nucleolin antibody. IP with No antibody (No

ab) and preimmune serum (IgG) were used as control.



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absence of NCL-K88ac in nucleolar structure as observed by

immunofluorescence.

If NCL-K88acis notinvolvedin RNAPI regulation, then what could

be itsfunction?Interestingly,it wasfoundthat nucleolin in thepres-

ence of PCAF enhanced IRF-2-dependant H4 promoter activity [23].

Recruitment of nucleolin to acetylated IRF-2 is requiredfor this pro-

moter activity. However, since we show in this manuscript that

nucleolin is also a substrate of PCAF, it would be interesting to

reevaluate the role of nucleolin acetylation in this transcriptional

regulation. NPM1 that share many functional similarities with

nucleolin [24] is also acetylated by p300 [25]. Acetytlated-NPM1 is

predominantly localized in the nucleoplasm [26] and has an en-hanced ability to activate transcription from chromatin templates.

However, we were unable to see any colocalization for NCL-K88ac

with transcriptionally active RNA polymerase II or any activation

of transcription on chromatin template in vitro (data not shown).

It is remarkable that NCL-K88ac distribution in the nucleoplasm

is co-localized with the nuclear domains enriched in splicing factor

SC35 and with the snRNP marker Y12 (Fig. 4). These nuclear speck-

les (also called interchromatin granule clusters, IGC) are very dy-

namic structures [27] and are also enriched in many other mRNA

splicing factors, RNA polymerase II subunits and diverse

transcription factors [28] but, transcription and pre-mRNA splicing

do not seem to take place within these structures [29]. The

association of NCL-K88ac with these speckles suggests two possi-ble functions for NCL-K88ac. First, it was shown that the

WB: AcNcl1

WB: H3 ac-lysine 14

WB: NCL

He

La

Inpu

t

Na

Bu

trea

ted

A

D

NaBu

Treated

Control

AcNcl1 NCL AcNcl1+NCL Merge DAPI

C

E

Re

lativep

roteinleve

l

0

0.5

1

1.5

2

2.5

Control NaBu

AcNcl

AcH3

0

100

200

300

400

500600

700

800

Ace

tylatednuc

leolinleve

l

B

NCL AcNcl1 NCL+AcNcl1 Merge DAPI

1 2

Fig. 3. NCL-K88ac is present in nucleoplasm and is excluded from nucleoli. (A) HeLa cells were stained with nucleolin antibody 5567 (red) andwith AcNcl1antibody(green).

DNA (blue)was counterstained with DAPI. Scale bar, 5 lm. (B) Nucleolin is hyperacetylated after NaBu treatment. Cell lysate from HeLa cells under normal growth conditions(lane 1) and after treatment with deacetylase inhibitors (5 mM sodium butyrate for 24 h) (lane 2) were analyzed on a 10% SDSPAGE, and analyzed by Western blot with

AcNcl1, H3 acetyl lysine 14 and nucleolin (NCL) antibodies. (C) Quantification of the Western blot data. (D) Immunofluorescence staining of control and cells treated with

deacetylase inhibitors (5 mM sodium butyrate) with nucleolin antibody coupled to Alexa Fluor 555 (molecular probes) (red) and with AcNcl1 antibody (green). DNA was

counterstained with DAPI (blue). Scale bar, 5 lm. (E) Quantification of immunofluorescence data from (D).



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Pearsons

Coe

fficien

t

0

0.2

0.4

0.6

0.8

1

SC35 andAcNcl

Y-12 andAcNcl

coilin andAcNcl

Men

dersC

oe

fficien

t

0

0.2

0.4

0.6

0.8

1

1.2

AcNcl toSC35

AcNcl toY-12

AcNcl tocoilin

SC35 toAcNcl

Y-12 toAcNcl

coilin toAcNcl

D E

C AcNcl1 coilin DAPIAcNcl1+coilin Merge

r = 0.297

AAcNcl1 SC35 DAPIMergeAcNcl1+SC35

r = 0.889

BAcNcl1 Y-12 DAPIMergeAcNcl1+Y-12

r = 0.761

Fig. 4. Immunofluorescence of NCL-K88ac and different nuclear markers. (A) Immunofluorescence staining of AcNcl1 (green) with nuclear markers SC35 (red), (B) Y-12 (red),

(C) Coilin (red) in formaldehyde fixed HeLa cells. DNA staining (blue) was counterstained with DAPI. Scale bar, 5 lm. Lower panels of each section (A, B, C) represent theenlargements represented by the squares in the corresponding upper images (scale bar 1 lm). The lower right insert of each panel correspond to the cytofluorogram whichwas used to determine the Pearsons coefficient (D) Histogram showing Pearsons coefficient for 15 individual cells analyzed in 3D for SC35 and AcNcl, Y-12 and AcNcl, coilin

and AcNcl. (E) Histogram showing Menders coefficient for 15 individual cells analyzed in 3D for SC35 and AcNcl, Y-12 and AcNcl, coilin and AcNcl.





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serine-2-phosphorylated form of the RNA polymerase II large sub-

unit, which is involved in transcription elongation was co-localized

with nuclear speckles [30]. As nucleolin has been involved in the

transcription of several Pol II genes and in transcription elongation

[5] it is also possible that the presence of NCL-K88ac in the speck-

les participates to the formation of transcription elongation com-

plexes for the nearby genes.

The second possibility is that nucleolin participates in the splic-

ing of some mRNAs. There are many examples of interactions of

nucleolin with different mRNAs with different effects on mRNA

stability and translation. Recently, a pull down assay followed by

microarray analysis identified several hundred of potential nucleo-lin mRNA targets [31]. Although the nucleolin interactome is not

known, several reports describe the interaction of nucleolin with

proteins involved in pre-mRNA splicing in particular, different

hnRNPs [32]. Our results also show that SC35 complexes contain

NCL-K88ac. Recently, RNP complexes formed on a specific HIV

pre-mRNA splicing site (SLS2-A7 RNA transcripts) in HeLa cell nu-

clear extracts identified hnRNP A1, nucleolin, hnRNP H and hnRNP

K that directly interact with SLS2-A7 RNA [33]. Nucleolin binds to a

cluster of successive canonical nucleolin recognition element (NRE

motifs) [21] in SLS2-A7 RNA. The authors showed a strong effect of

hnRNP K on HIV-1 alternative splicing, but they have not tested the

effect of the interaction of nucleolin with HIV-1 mRNA. This opens

the possibility that the interaction of nucleolin with this RNA af-fects its alternative splicing.

He

La

U87

Non-s

timu

lated

PBMC

Stimu

lated

PBMC

LNcap

Caco-2

Ramos

Ra

Ji

Dau

di

WB: Ncl

WB: beta-actin

WB:AcNcl1

A

C

B

Ra

tioo

fAc

Nc

l/Ncl

1 2 3 4 5 6 7 8 9

0

0.005

0.01

0.015

0.02

0.025

AcNcl1 SC35 AcNcl1+SC35 Merge DAPI

Fig. 5. Acetylation of nucleolin in different cell lines. (A) Cell lysates were prepared from different cell lines as indicated on the figure (lanes 19) and immunoblotted with

AcNcl1, nucleolinand b-actin antibodies. (B) Quantification of Western blot results showing the ratio of NCL-K88ac versus total nucleolin. (C) Immunofluorescence staining of

nucleolin in stimulated PBMC cells using AcNcl1 (green) and SC35 antibodies (red). DNA was stained with DAPI (blue). Scale bar: 5 lm.





8/8

The characterization and functional significance of nucleolin

post-translational modifications are still unexplored. In this work,

we provide evidence that nucleolin is acetylated in vivo and this

modification drastically changes its cellular localization. The pres-

ence of NCL-K88ac in nuclear speckles suggests that this nucleolin

pool may be involved in pre-mRNA synthesis or metabolism. The

specific antibodies developed against NCL-K88ac herein, should

be useful to explore this novel nucleolin function.

Acknowledgements

We thank PLATIM (PLAteau Technique dImagerie et de Micros-

copie, UMS3444, Lyon, FRANCE) for access to microscopy facility.

T.K.K. is a recipient of Sir J.C. Bose National Fellowship from

Department of Science and Technology, Government of India. This

work was supported by grants from Agence Nationale de la Recher-

che (ANR-07-BLAN-0062-01), Rgion Rhne-Alpes MIRA 2010,

Association pour la Recherche sur le Cancer No. ECL2010R01122,

CEFIPRA No. 3803-1, CNRS and Ecole Normale Suprieure de Lyon.

S.D. was supported by CEFIPRA and by Fondation pour la Recherche

Mdicale (FRM).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.febslet.2013.01.

035.

References

[1] Orrick, L.R., Olson, M.O. and Busch, H. (1973) Comparison of nucleolar proteins

of normal rat liver and Novikoff hepatoma ascites cells by two-dimensional

polyacrylamide gel electrophoresis. Proc. Natl. Acad. Sci. USA 70, 13161320.

[2] Ginisty, H.,Sicard, H.,Roger, B. andBouvet, P. (1999) Structure andfunctions of

nucleolin. J. Cell Sci. 112 (Pt 6), 761772.

[3] Ginisty, H., Amalric, F. and Bouvet, P. (1998) Nucleolin functions in the first

step of ribosomal RNA processing. EMBO J. 17, 14761486.

[4] Bouvet, P., Diaz, J.J., Kindbeiter, K., Madjar, J.J. and Amalric, F. (1998) Nucleolin

interacts with several ribosomal proteins through its RGG domain. J. Biol.Chem. 273, 1902519029.

[5] Cong, R., Das, S. and Bouvet, P. (2011) The multiple properties and functions of

nucleolin in: The Nucleolus Protein Reviews (Olson, M.O.J., Ed.), pp. 185212,

Springer-Verlag New York Inc.

[6] Storck, S., Thiry, M. and Bouvet, P. (2009) Conditional knockout of nucleolin in

DT40 cells reveals the functional redundancy of its RNA-binding domains. Biol.

Cell 101, 153167.

[7] Rickards, B., Flint, S.J., Cole, M.D. and LeRoy, G. (2007) Nucleolin is required for

RNA polymerase I transcription in vivo. Mol. Cell. Biol. 27, 937948.

[8] Ugrinova, I., Monier, K., Ivaldi, C., Thiry, M., Storck, S., Mongelard, F. and

Bouvet, P. (2007) Inactivation of nucleolin leads to nucleolar disruption, cell

cycle arrest and defects in centrosome duplication. BMC Mol. Biol. 8, 66.

[9] Angelov, D. et al. (2006) Nucleolin is a histone chaperone with FACT-like

activity and assists remodeling of nucleosomes. EMBO J. 25, 16691679.

[10] Cong, R., Das, S., Ugrinova, I., Kumar, S., Mongelard, F., Wong, J. and Bouvet, P.

(2012) Interaction of nucleolin with ribosomal RNA genes and its role in RNA

polymerase I transcription. Nucleic Acids Res. 40, 94419454.

[11] Belenguer, P., Caizergues-Ferrer, M., Labbe, J.C., Doree, M. and Amalric, F.(1990) Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein

kinase. Mol. Cell. Biol. 10, 36073618.

[12] Borer, R.A., Lehner, C.F., Eppenberger, H.M. and Nigg, E.A. (1989) Major

nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56, 379390.

[13] Bouche, G., Caizergues-Ferrer, M., Bugler, B. and Amalric, F. (1984)

Interrelations between the maturation of a 100 kDa nucleolar protein and

pre rRNA synthesis in CHO cells. Nucleic Acids Res. 12, 30253035.

[14] Tediose, T., Kolev, M., Sivasankar, B., Brennan, P., Morgan, B.P. and Donev, R.

(2010) Interplay between REST and nucleolin transcription factors: a key

mechanism in the overexpression of genes upon increased phosphorylation.

Nucleic Acids Res. 38, 27992812.

[15] Yang, C., Maiguel, D.A. and Carrier, F. (2002) Identification of nucleolin and

nucleophosmin as genotoxic stress-responsive RNA-binding proteins. NucleicAcids Res. 30, 22512260.

[16] Lischwe, M.A., Roberts, K.D., Yeoman, L.C. and Busch, H. (1982) Nucleolar

specific acidic phosphoprotein C23 is highly methylated. J. Biol. Chem. 257,

46004602.

[17] Raman, B. et al. (2001) N(omega)-arginine dimethylation modulates the

interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic

acids. Nucleic Acids Res. 29, 33773384.

[18] Salazar, R., Brandt, R., Kellermann, J. and Krantz, S. (2000) Purification and

characterization of a 200 kDa fructosyllysine-specific binding protein fromcell

membranes of U937 cells. Glycoconj. J. 17, 713716.

[19] Sterner, D.E. and Berger, S.L. (2000) Acetylation of histones and transcription-

related factors. Microbiol. Mol. Biol. Rev. 64, 435459.

[20] Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C.,

Olsen, J.V. and Mann, M. (2009) Lysine acetylation targets protein complexes

and co-regulates major cellular functions. Science 325, 834840.

[21] Ghisolfi-Nieto, L., Joseph, G., Puvion-Dutilleul, F., Amalric, F. and Bouvet, P.

(1996) Nucleolin is a sequence-specific RNA-binding protein: characterization

of targets on pre-ribosomal RNA. J. Mol. Biol. 260, 3453.

[22] Zentner, G.E., Saiakhova, A., Manaenkov, P., Adams, M.D. and Scacheri, P.C.

(2011) Integrative genomic analysis of human ribosomal DNA. Nucleic Acids

Res. 39, 49494960.

[23] Masumi, A., Fukazawa, H., Shimazu, T., Yoshida, M., Ozato, K., Komuro, K. and

Yamaguchi, K. (2006) Nucleolin is involved in interferon regulatory factor-2-

dependent transcriptional activation. Oncogene 25, 51135124.

[24] Lindstrom, M.S. (2011) NPM1/B23: a multifunctional chaperone in

ribosome biogenesis and chromatin remodeling. Biochem. Res. Int. 2011,

195209.

[25] Swaminathan, V., Kishore, A.H., Febitha, K.K. and Kundu, T.K. (2005) Human

histone chaperone nucleophosmin enhances acetylation-dependent

chromatin transcription. Mol. Cell. Biol. 25, 75347545.

[26] Shandilya, J., Swaminathan, V., Gadad, S.S., Choudhari, R., Kodaganur, G.S. and

Kundu, T.K. (2009) Acetylated NPM1 localizes in the nucleoplasm and

regulates transcriptional activation of genes implicated in oral cancer

manifestation. Mol. Cell. Biol. 29, 51155127.

[27] Lamond, A.I. and Spector, D.L. (2003) Nuclear speckles: a model for nuclear

organelles. Nat. Rev. Mol. Cell. Biol. 4, 605612.

[28] Saitoh, N., Spahr, C.S., Patterson, S.D., Bubulya, P., Neuwald, A.F. and Spector,D.L. (2004) Proteomic analysis of interchromatin granule clusters. Mol. Biol.

Cell 15, 38763890.

[29] Misteli, T. and Spector, D.L. (1999) RNA polymerase II targets pre-mRNA

splicing factors to transcription sites in vivo. Mol. Cell 3, 697705.

[30] Mortillaro, M.J., Blencowe, B.J., Wei, X., Nakayasu, H., Du, L., Warren, S.L.,

Sharp, P.A. and Berezney, R. (1996) A hyperphosphorylated form of the large

subunit of RNA polymerase II is associated with splicing complexes and the

nuclear matrix. Proc. Natl. Acad. Sci. USA 93, 82538257.

[31] Abdelmohsen, K. et al. (2011) Enhanced translation by nucleolin via G-rich

elements in coding and non-coding regions of target mRNAs. Nucleic Acids

Res. 39, 85138530.

[32] Uribe, D.J., Guo, K., Shin, Y.J. and Sun, D. (2011) Heterogeneous nuclear

ribonucleoprotein K and nucleolin as transcriptional activators of the vascular

endothelial growth factor promoter through interaction with secondary DNA

structures. Biochemistry 50, 37963806.

[33] Marchand, V. et al. (2011) Identification of protein partners of the human

immunodeficiencyvirus1 tat/rev exon 3 leads to the discovery of a new HIV-1

splicing regulator, protein hnRNP K. RNA Biol. 8, 325342.



li i f t FEBS L tt (2013) htt //d d i /10 1016/j f b l t 2013 01 035
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