Identification of protein factors and U3 snoRNAs from a Brassica oleracea RNP complex involved in the processing of pre-rRNA

Identification of protein factors and U3 snoRNAs froma Brassica oleracea RNP complex involved in the processingof pre-rRNA

Hala Samaha1,†, Valerie Delorme1, Frederic Pontvianne1,‡, Richard Cooke1, Francois Delalande2, Alain Van Dorsselaer2,

Manuel Echeverria1 and Julio Saez-Vasquez1,*

1Laboratoire Genome et Developpement des Plantes, UMR 5096 CNRS-IRD-UPVD; Perpignan France. 52 av.¨Paul Alduy, 66860

Perpignan-Cedex, France, and2Laboratoire de Spectrometrie de Masse Bio-Organique Departement des Sciences Analytiques, Institut Pluridisciplinaire

Hubert Curien, UMR 7178 (CNRS-ULP) ECPM, 25 rue Becquerel F67087-Strasbourg-Cedex, France

Received 1 July 2009; revised 18 September 2009; accepted 15 October 2009; published online 8 December 2009.*For correspondence (fax +33 4 68668499; e-mail [email protected]).†Present address: Universite de Pircadie Jules Verne, Amiens, France.‡Present address: Washington University, St. Louis, MO, USA.

SUMMARY

We report on the structural characterization of a functional U3 snoRNA ribonucleoprotein complex isolated

from Brassica oleracea. The BoU3 snoRNP complex (formerly NF D) binds ribosomal DNA (rDNA), specifically

cleaves pre-rRNA at the primary cleavage site in vitro and probably links transcription to early pre-rRNA

processing in vivo. Using a proteomic approach we have identified 62 proteins in the purified BoU3 snoRNP

fraction, including small RNA associated proteins (Fibrillarin, NOP5/Nop58p, Diskerin/Cbf5p, SUS2/PRP8 and

CLO/GFA1/sn114p) and 40S ribosomal associated proteins (22 RPS and four ARCA-like proteins). Another

major protein group is composed of chaperones/chaperonins (HSP81/TCP-1) and at least one proteasome

subunit (RPN1a). Remarkably, RNA-dependent RNA polymerase (RdRP) and Tudor staphylococcal nuclease

(TSN) proteins, which have RNA- and/or DNA-associated activities, were also revealed in the complex.

Furthermore, three U3 snoRNA variants were identified in the BoU3 snoRNP fraction, notably an evolutionarily

conserved and variable stem loop structure located just downstream from the C-box domain of the U3

sequence structures. We conclude that the BoU3 snoRNP complex is mainly required for 40S pre-ribosome

synthesis. It is also expected that U3 snoRNA variants and interacting proteins might play a major role in

BoU3 snoRNP complex assembly and/or function. This study provides a basis for further investigation of these

novel ribonucleoprotein factors and their role in plant ribosome biogenesis.

Keywords: U3 snoRNP, pre-rRNA, processing, ribosome, Arabidopsis, proteomic.

INTRODUCTION

Ribosome biogenesis requires coordination between

transcription and processing of ribosomal RNA precursors

(pre-rRNA), import–export of ribosomal proteins (r proteins)

and assembly and transport of ribosome particles (Grandi

et al., 2002; Schafer et al., 2003; Tschochner and Hurt, 2003).

All these processes begin in the nucleolus with 90S pre-

ribosome particle formation, and end in the cytoplasm after

export and final maturation of 60S and 40S subunits. One

way to coordinate these complex reactions is to concentrate

the factors involved in the ribosome biogenesis process in a

multifunctional protein complex (reviewed in Fatica and

Tollervey, 2002; Fromont-Racine et al., 2003).

In the nucleolus, transcription of 18S, 5.8S and 25S–28S

rRNA genes by RNA polymerase I (RNA polI) is linked to

processing of the primary transcript (pre-rRNA) and 90S pre-

ribosomal particle synthesis. Through transcription of the

full-length pre-rRNA, the external (5¢ ETS and 3¢ ETS) and

internal (ITS-1 and ITS-2) spacers are removed to generate

mature rRNA (Nomura, 2001; Saez-Vasquez and Echeverria,

2006). Likewise, modification of numerous rRNA residues

occurs. During this process, numerous multifunctional

factors associate with the nascent transcribed pre-rRNA,

including small nucleolar RNA (snoRNA) as well as ribo-

somal and non-ribosomal proteins. The latter include endo

ª 2009 The Authors 383Journal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2010) 61, 383–398 doi: 10.1111/j.1365-313X.2009.04061.x

and exonucleases that process the pre-rRNA, pseudouridine

synthases and methyltransferases that catalyse pseudou-

ridylation and 2¢-O-ribose methylation of numerous rRNA

residues, and helicases and chaperones that might facilitate

folding and cleavage of RNA (Eichler and Craig, 1994;

Venema and Tollervey, 1995; Kufel et al., 1999; Barneche

et al., 2000; Comella et al., 2008).

The recent development of sensitive methods for identi-

fying proteins by mass spectrometry has largely contributed

to our knowledge of ribosome formation pathways. Thus,

biochemical characterization of the 90S pre-ribosome parti-

cle revealed that this complex contains mainly ribosomal

proteins from the 40S subunit and �35 non-ribosomal

components, including proteins associated with U3 sno-

RNA, in addition to many others factors required for 18S

rRNA synthesis (Grandi et al., 2002; Schafer et al., 2003).

Interestingly, it almost completely lacks components from

the 60S subunit (Grandi et al., 2002). Thus, the 40S synthesis

machinery is associated with the larger pre-rRNA forms,

whereas factors required for 60S formation bind later,

indicating a clear separation between 40S and 60S biosyn-

thesis pathways. A few years ago, a large U3 snoRNP

complex named the SSU processome was isolated from

Saccharomyces cerevisiae (Dragon et al., 2002). This com-

plex is required for the processing of 18S rRNA biogenesis. It

contains the U3 snoRNA, 17 U3 proteins (Utp1–17), 10

U3 snoRNA-associated proteins, Rrp5 protein and five small

subunit ribosomal proteins (Dragon et al., 2002). Further

genetic dissection of the yeast U3 snoRNP identified the Utp

complex, which is formed in the absence of U3 and interacts

directly with the rDNA 5¢ ETS region just before the assem-

bly of U3 snoRNP with the pre-rRNA (Gallagher et al., 2004).

Several pre-rRNA endonucleolytic cleavage events in the

nucleolus require U3 snoRNA and lead selectively to the

generation of 18S rRNA (Hughes and Ares, 1991; Beltrame

and Tollervey, 1992, 1995). The U3 RNA contains the

consensus C and D box, and is thus classified in the C/D

box family of snoRNA. It therefore associates with the

common C/D snoRNP proteins like Snu13p, Nop56p, Nop58

and Nop1/fibrillarin (Hartshorne and Agabian, 1994; Lafon-

taine and Tollervey, 1999; Lukowiak et al., 2000; Granneman

et al., 2002; Leary et al., 2004; Clery et al., 2007). The

U3 snoRNP is the only C/D snoRNP for which specific

components have been identified. Indeed, functional and

structural analysis of U3 sequences from diverse organisms

revealed at least five additional short elements, known as

boxes GAC A, A¢, B and C¢. Several reports have clearly

demonstrated that the C/D boxes are required for the

nucleolar localization of U3 snoRNA, whereas the GAC A

and A¢ boxes are essential for directing the specific 18S

pre-rRNA cleavage (Speckmann et al., 1999; Borovjagin and

Gerbi, 2001).

In plants, little is known about the factors involved in

ribosome biosynthesis. Proteomic approaches have been

successfully used to identify ribosomal proteins from the

purified cytosolic ribosome (Giavalisco et al., 2005; Zanetti

et al., 2005), as well as nucleolar proteins in Arabidopsis

(Pendle et al., 2005). In total, 217 nucleolar proteins were

identified in Arabidopsis, including fibrillarin, Nop56, Nop58

and some U3-specific proteins (Pendle et al., 2005). How-

ever, this number is relatively low compared with approx-

imately 4500 proteins identified in the human nucleolus

(Leung et al., 2006; Ahmad et al., 2009), suggesting the

existence of other stable and/or transitory nucleolar proteins

in plants. On the other hand, hundreds of C/D box snoRNAs

have been reported in plants, including U3 snoRNA (Leader

et al., 1997; Barneche et al., 2001; Brown et al., 2003a,b;

Chen et al., 2003), which can be folded into a secondary

structure similar to that from yeast, trypanosome and

xenopus (but not to U3 sequences from other vertebrates)

(Kiss and Solymosy, 1990; Marshallsay et al., 1990). How-

ever, plant U3 sequences display particular features with

respect to U3 snoRNA from yeast and metazoans (Kiss and

Solymosy, 1990; Marshallsay et al., 1990). Indeed, although

the U3 snoRNA structures from plants and other organisms

are conserved, the sequence identity between plant

U3 snoRNA and those of human, xenopus, trypanosome

and yeast is less than 35%. In contrast, sequence identity

between Brassica, Arabidopsis, rice (Oryza sativa), wheat

(Triticum spp.), maize (Zea mays), Solanum and Lotus

japonica (see below for secondary structures) is greater

than 66%. Among all potential U3 snoRNA proteins

described in plants (reviewed recently in Saez-Vasquez and

Medina, 2008), fibrillarin is the best characterized (Pih et al.,

2000; Barneche et al., 2001). Interestingly, the homologues

of U3 nucleolar proteins, such as Nop56p, Nop58 and

fibrillarin, were identified in the Arabidopsis nuclear matrix,

together with nucleolin, histone deacetylase, translation

factor eIF-1, two chaperones and other non-nucleolar or

ribosome-related proteins (Calikowski et al., 2003). Never-

theless, in the absence of any experimentation it would be

premature to propose any functional relationship between

U3 snoRNP and other non-nucleolar proteins.

Previously, a large U3 snoRNP (NFD, renamed in this

paper as BoU3 snoRNP) was described in our laboratory

after its purification from cauliflower (Brassica oleracea var.

botrytis) inflorescence. It was characterized by its capacity to

bind pre-rRNA and accurately produce the first cleavage of

the pre-rRNA in vitro (Saez-Vasquez et al., 2004a,b). Inter-

estingly, the Brassica U3 snoRNP also binds to rDNA in vitro.

Indeed, the BoU3 snoRNP complex was purified based on its

sequence-specific rDNA binding activity to a double-

stranded rDNA cluster motif, A123B (see Figure 1b), located

just upstream from the primary cleavage site in the pre-RNA

5¢ ETS (Caparros-Ruiz et al., 1997). Thus, the BoU3 snoRNP

most probably links RNA polI transcription to processing

and maturation of pre-RNA. This might occur by a mecha-

nism in which the U3 snoRNP processing complex first

384 Hala Samaha et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 383–398

assembles on rDNA, and subsequently interacts with the

nascent pre-rRNA produced during RNA polI transcription

(Saez-Vasquez et al., 2004b).

In this article, using a proteomic approach and a cloning

strategy, we identified 62 proteins as well as three phyloge-

netically conserved U3 snoRNA variant sequences in the

purified BoU3 snoRNP fraction. The potential role of these

RNA protein factors in ribosome biosynthesis in addition to

RNP complex assembly is discussed.

RESULTS

Purification of a large quantity of BoU3 snoRNP

Previously, we showed that BoU3 snoRNP activity (defined

by its ability to bind rDNA and cleave pre-rRNA at the P site)

can be obtained from cauliflower inflorescence and subse-

quently purified by sequential chromatography (Saez-

Vasquez et al., 2004b). Here, in order to purify a large quantity

of U3 snoRNP for protein sequencing, the nuclear extracts

were fractionated using DEAE Sepharose, Heparin Sepha-

rose and oliA DNA Sepharose (Figure 1a). Because NFB

(a subcomplex of BoU3 snoRNP) dissociates during frac-

tionation on Sephacryl-300 (Saez-Vasquez et al., 2004b), we

decided to omit this purification step to recover a greater

number of protein factors associated with BoU3 snoRNP.

As shown in Figure 1(b), most of the BoU3 snoRNP activity

binds to the oliA column at 100 mM KCl (lane 2, flow-

through fraction), and can subsequently be recovered after

an elution step at 350 mM KCl. Neither BoU3 snoRNP nor

NFB activity was detected in the 1000 mM KCl fraction

(lanes 8–12), indicating that most of the activity was eluted

with 350 mM KCl (lanes 3–7). As observed previously, much

of the BoU3 snoRNP complex bound to the column was

dissociated into NFB complexes after elution. The greater

part of the NFB activity from the Heparin 600 fraction does

not bind oliA Sepharose (lane 2). In agreement with this

observation, footprinting analysis shows that BoU3 snoRNP

binds specifically to the A123 boxes, whereas NFB binds to

the B box (Caparros-Ruiz et al., 1997). To visualize the pro-

teins eluted at 350 mM KCl from the oliA column, fractions

were concentrated, separated by SDS-PAGE and visualized

by SyproRuby. This fraction reveals at least 30 major pro-

teins ranging from�14 to �200 kDa (Figure 1c). This protein

band pattern is relatively similar to that observed by silver

staining reported previously (Saez-Vasquez et al., 2004b).

The differences between the two profiles are probably

caused by the reactivity of the proteins with different stain-

ing components.

Mass spectrometry analysis of proteins isolated

from 1D SDS-PAGE

To identify proteins co-purified in the BoU3 snoRNP frac-

tion, the gel was sliced and submitted to mass spectrometry

analysis. A total of 466 peptide sequences, representing 62

unique proteins, were identified (Table 1). As expected,

fibrillarin and NOP5/Nop58-like proteins were detected,

together with 27 ribosomal proteins (RPs). Among the RPs,

22 belong to the small ribosome subunit (40S RPS) and five

belong to the large ribosome subunit (60S RPL). Two other

RPs are related to ribosome function (ARCA and eIF3H1).

Interestingly, at least two additional small nuclear U5 snRNP

(SUS/Prp8 and CLO/GFA1/Snu114p) and one H/ACA

snoRNP protein (Dyskerin/Cbf5p) were identified in the

BoU3 snoRNP fraction. One group of proteins, including one

chaperone (HSP81), eight chaperonins from the TCP1 com-

plex and one regulatory subunit from the 26S proteasome

(RPN1a) can be associated with one of the multiple ribosome

biogenesis steps. Six polypeptides are related to DNA and/or

RNA metabolism (three RNA polII subunits, one DNA gyrase

subunit, one RNA-dependent RNA polymerase and a Tudor

Cauliflower

Whole cell extract

DEAE

NH4(SO4)2fraction

–

BoU3snoRNP

NF B

Heparine

FT-175

FT-100

E-600

FT-100 E-1000E-350

OliA DNA

FT E-350 E-1000

E-350

1 2 3 4 5 6 7 8 9 10 11 12

A1 A2 A3 B P 18S

103 741+1

185

TIS

205

E-350

116

97

200

66

45

31

2121

14

(a) (b)

(c)

Figure 1. Purification of BoU3 snoRNP from cauliflower whole-cell extracts

(Brassica oleracea var. botrytis).

(a) Scheme for sequential chromatography using DEAE-Sepharose CL-6B,

Heparin-Sepharose and oliA DNA Sepharose.

(b) Analysis of BoU3 snoRNP activity in the oliA eluted fractions using an

electrophoresis mobility shift assay (EMSA). The rDNA A123BP probe (see

diagram) was incubated with 15 ll of fraction FT (lane 2), eluted with 350 mM

KCl (lanes 3–7) or eluted with 1000 mM KCl (lanes 8–12). Control rDNA probe

alone (lane 1). The arrow shows the rDNA BoU3 snoRNP protein complex and

the light-black arrow the rDNA-NFB protein subcomplexes.

(c) OliA fraction subjected to electrophoresis on an 8.5% SDS/polyacrylamide

gel and visualized by Sypro Ruby staining. The EMSA probe shown at the

bottom includes the 5¢ ETS sequences from +103 to +205 bp.

Brassica oleracea U3 snoRNP 385


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IHIQ

DS

TG

HE

FAT

R,

LGG

AFA

PK

,LT

IIEE

AR

,Y

ALT

YR

,Y

PD

PLI

KP

ND

TIK

At5

g07

090

40S

rib

oso

mal

pro

tein

S5-

2P

5142

7|R

S52

_AR

AT

H22

098.

83

18.7

0Q

AV

DIS

PLR

,T

IAE

CLA

DE

LIN

AA

K,

VN

QA

IFLL

TT

GA

RA

t3g

1194

0

40S

rib

oso

mal

pro

tein

S6-

1O

4854

9|R

S61

_AR

AT

H28

248,

31

6.02

LSQ

EV

SG

DA

LGE

EFK

No

PA

t4g

3170

040

Sri

bo

som

alp

rote

inS

7Q

9XH

45|R

S7_

BR

AO

L22

135.

93

22.5

0FS

GN

DV

IFV

AT

R,

LET

MV

GV

YR

,T

LTS

VH

EA

MLE

DV

AY

PA

EIV

GK

40S

rib

oso

mal

pro

tein

S9-

1Q

9LX

G1|

RS

91_A

RA

TH

2301

9.4

727

.30

DLL

TLD

EK

,IF

EG

EA

LLR

,LQ

TIV

FK,

LVG

EY

GLR

,Q

LVN

IPS

FMV

R,

RLQ

TIV

FK,

YG

LLD

ES

QN

K

No

PA

t5g

1520

0

40S

rib

oso

mal

pro

tein

Sa-

1Q

0868

2|R

SS

A1_

AR

AT

H31

963.

57

30.3

0FA

QY

TG

AN

AIA

GR

,FV

DIG

IPA

NN

K,

HT

PG

TFT

NQ

MQ

TS

FSE

PR

,LL

ILT

DP

R,

ND

GIY

IFN

LGK

,V

IVA

IEN

PQ

DIIV

QS

AR

,W

DV

MV

DLF

FYR

,

At1

g72

370

Rib

oso

mal

pro

tein

S8

Q0J

DZ

7|Q

0JD

Z7_

OR

YS

J24

911.

13

18.6

0LD

TG

NY

SW

GS

EA

VT

R,

SA

IVQ

VD

AA

PFK

,T

LDS

HIE

EQ

FGS

GR

Rib

oso

mal

pro

tein

S8

A9S

3Y5|

A9S

3Y5_

PH

YP

A24

321,

41

13.9

0V

LDV

VY

NA

SN

NE

LVR

,LD

TG

NY

SW

GS

EA

VT

R60

Sri

bo

som

alp

rote

ins

60S

acid

icri

bo

som

alp

rote

inP

0-1

O04

204|

RLA

01_A

RA

TH

2521

8.1

15.

46G

TV

EIIT

PV

ELI

KA

t2g

4001

060

Sri

bo

som

alp

rote

inL1

7-2

P51

413|

RL1

72_A

RA

TH

1954

6.9

15.

85S

AQ

FVLD

LLK

No

PA

t1g

6743

060

Sri

bo

som

alp

rote

inL1

8-2

P42

791|

RL1

82_A

RA

TH

2094

9.7

16.

95IA

VLV

GT

ITD

DLR

At3

g05

590

60S

rib

oso

mal

pro

tein

L5-2

P49

227|

RL5

2_A

RA

TH

3442

0.2

313

.30

ALL

DV

GLI

R,

DIV

AQ

IVS

AS

IAG

DIV

K,

GA

LDG

GLD

IPH

SD

KN

oP

At5

g39

740

60S

rib

oso

mal

pro

tein

L9-1

P49

209|

RL9

1_A

RA

TH

2198

7.6

15.

18FL

DG

LYV

SE

KN

oP

At1

g33

120

Ch

aper

on

es/p

rote

aso

me

26S

pro

teas

om

ere

gu

lato

rysu

bu

nit

S2

(RP

N1a

)Q

9SIV

2|Q

9SIV

2_A

RA

TH

9813

0.8

34.

26LS

EG

YLT

LAR

,V

GQ

AV

DV

VG

QA

GR

,Y

IPLS

PIL

EG

FIIL

KA

t2g

2058

0

Hea

tsh

ock

pro

tein

81-3

P51

818|

HS

P83

_AR

AT

H80

077.

02

3.00

AD

LVN

NLG

TIA

R,

AP

FDLF

DT

KA

t5g

5600

0T

-co

mp

lex

pro

tein

1su

bu

nit

alp

ha

P28

769|

TC

PA

_AR

AT

H59

198.

45

13.9

0E

QLA

IAE

FAD

ALL

IIPK

,E

VG

DG

TT

SV

VIV

AA

ELL

K,

MLV

DD

IGD

VT

ITN

DG

AT

ILR

,T

SLG

PV

GLD

K,

VLV

ELA

ELQ

DR

At3

g20

050

T-c

om

ple

xp

rote

in1,

bet

asu

bu

nit

Q94

0P8|

Q94

0P8_

AR

AT

H57

269.

63

11.2

0A

LVA

IPT

TIA

DN

AG

LDS

AE

LVA

QLR

,D

SFL

DE

GFI

LDK

,V

QD

DE

VG

DG

TT

SV

VV

LAG

ELL

R

At5

g20

890



Tab

le1

(Co

nti

nu

ed)

Pro

tein

nam

eP

rote

inac

cess

ion

nu

mb

ers

Pro

tein

mo

lecu

lar

wei

gh

t(D

a)

Nu

mb

ero

fu

niq

ue

pep

tid

es

Per

cen

tag

ese

qu

ence

cove

rag

eP

epti

des

Iden

tifi

edin

Ara

bid

op

sis

At_

AG

I

T-c

om

ple

xp

rote

in1,

bet

asu

bu

nit

A5C

537|

A5C

537_

VIT

VI

5730

6.1

19.

49Q

AV

LLS

AT

EA

AE

MIL

RT

-co

mp

lex

pro

tein

1,g

amm

asu

bu

nit

O81

503|

O81

503_

AR

AT

H60

323.

04

9.55

IDD

IVS

GIK

,LV

PG

GG

AT

ELT

VS

AT

LK,

TLA

QN

CG

VN

VIR

,W

PY

EA

AA

IAFE

AIP

RT

-co

mp

lex

pro

tein

1,d

elta

sub

un

itQ

8L99

4|Q

8L99

4_A

RA

TH

5755

6,7

24.

32A

TG

CN

VLL

IQK

,IA

VIQ

FQIS

PP

KT

-co

mp

lex

pro

tein

1,et

asu

bu

nit

Q9S

F16|

Q9S

F16_

AR

AT

H59

759.

712

27.3

0D

SFL

VD

GV

AFK

,FN

IFS

GC

PS

GR

,G

GA

DQ

FIE

EA

ER

,G

SV

TIS

ND

GA

TIM

K,

ILV

DIA

K,

INA

INA

AT

EA

AC

LILS

VD

ET

VK

,LA

IGD

LAT

QY

FAD

R,

LNLI

GIK

,S

LHD

AIM

IVR

,S

QD

SE

VG

DG

TT

TV

VLL

AA

EFL

K,

SQ

LFIN

SY

AK

,T

FSY

AG

FEQ

QP

K

At3

g11

830

T-c

om

ple

xp

rote

in1,

thet

asu

bu

nit

Q94

K05

|Q94

K05

_AR

AT

H56

933.

85

12.9

0G

ST

DS

ILD

DLE

R,

LFV

TN

DA

AT

IVN

ELE

IQH

PA

AK

,V

AV

FAG

GV

DT

TA

TE

TK

,V

WD

LFA

TK

,Y

AE

SFE

FVP

K

At3

g03

960

T-c

om

ple

xp

rote

in1,

zeta

sub

un

itQ

94E

Z9|

Q94

EZ

9_A

RA

TH

5893

2.5

1130

.80

AQ

LGV

EA

FAN

ALL

VV

PK

,G

IDP

PS

LDLL

AR

,LV

EG

LVLD

HG

SR

,LY

EG

LAD

QLT

DIV

VN

SV

LCIR

,Q

LIN

SG

PV

IAS

QLL

LVD

EV

IR,

SE

INA

GFF

YS

NA

EQ

R,

TLA

EN

AG

LDT

QD

VIIS

LTS

EH

DK

,T

PV

VM

GD

EP

DK

EIL

K,

VLN

PN

AE

VLN

K,V

LVD

GFE

IAK

,YT

FVE

QV

KT

-co

mp

lex

pro

tein

1,su

bu

nit

epsi

lon

O04

450|

TC

PE

_AR

AT

H51

129.

35

16.3

0A

FAE

ALD

SV

PM

ALA

EN

SG

LQP

IET

LSA

VK

,A

VLA

VA

DLE

R,

DV

NLD

LIK

,E

QN

VFE

TLI

GK

,W

VG

GV

ELE

LIA

IAT

GG

R

At1

g24

510

Rib

oso

me

fun

ctio

nT

ran

slat

ion

init

iati

on

fact

or

3su

bu

nit

HQ

9C5Z

2|E

IF3H

_AR

AT

H38

355.

31

3.26

VV

QIE

GLA

VLK

At1

g10

840

GLP

-bin

din

gp

rote

in1a

(Fra

gm

ent)

Q84

V99

|Q84

V99

_AR

AT

H5

759.

32

41.8

0A

AFL

TN

DY

TK

,LT

AT

ELS

TY

AT

NK

AR

CA

/RA

CK

1AR

EC

EP

TO

RFO

RA

CT

IVA

TE

DC

KIN

AS

E1

A(B

elo

ng

sto

the

WD

rep

eat

Gp

rote

inb

eta

fam

ily)

O24

456|

GB

LP_A

RA

TH

3572

9.9

218

.00

DG

VV

LLW

DLA

EG

K,

DV

LSV

AFS

LDN

R,

FSP

NT

LQP

TIV

SA

SW

DK

,LW

DLA

AG

VS

TR

,S

IILW

K

At1

g18

080

Gu

anin

en

ucl

eoti

de-

bin

din

gp

rote

insu

bu

nit

bet

a-lik

ep

rote

in(B

elo

ng

sto

the

WD

rep

eat

Gp

rote

inb

eta

fam

ily)

Q39

336|

GB

LP_B

RA

NA

3570

5.6

522

.00

AH

TD

MV

TA

IAT

PID

NS

DT

IVS

AS

R,

DG

VV

LLW

DLA

EG

K,

DV

LSV

AFS

LDN

R,

LWD

LAA

GV

ST

R,

YW

LCA

AT

EQ

GIK

Gu

anin

en

ucl

eoti

de-

bin

din

gp

rote

insu

bu

nit

bet

a-lik

ep

rote

in(A

RC

A,

Trp

-Asp

40-a

are

pea

ts)

P49

026|

GB

LP_T

OB

AC

2861

1.9

114

.20

DV

LSV

AFS

VD

NR

,D

GV

ILLW

DLA

EG

K



Tab

le1

(Co

nti

nu

ed)

Pro

tein

nam

eP

rote

inac

cess

ion

nu

mb

ers

Pro

tein

mo

lecu

lar

wei

gh

t(D

a)

Nu

mb

ero

fu

niq

ue

pep

tid

es

Per

cen

tag

ese

qu

ence

cove

rag

eP

epti

des

Iden

tifi

edin

Ara

bid

op

sis

At_

AG

I

Pu

tati

veu

nch

arac

teri

zed

pro

tein

WD

40d

om

ain

(Po

pu

lus

tric

ho

carp

a)A

9P92

6|A

9P92

6_P

OP

TR

3587

2.0

112

.80

DG

VIL

LWD

LAE

GK

,FS

PN

TLQ

PT

IVS

AS

WD

K

DN

A/R

NA

rela

ted

acti

viti

esT

ud

or

do

mai

n-c

on

tain

ing

pro

tein

(TS

N)

Q8V

ZG

7|Q

8VZ

G7_

AR

AT

H11

466

6.8

33.

81D

LGLE

LVE

NG

LAK

,IP

AV

VE

YV

LSG

HR

,T

NV

AT

VLL

EA

GLA

KA

t5g

0735

0

RN

A-d

irec

ted

RN

Ap

oly

mer

ase

O82

189|

O82

189_

AR

AT

H10

263

7.3

10.

88Y

GV

FFP

QK

At2

g19

920

DN

A-d

irec

ted

RN

Ap

oly

mer

ase

IIsu

bu

nit

RP

B1

P18

616|

RP

B1_

AR

AT

H27

721.

71

4.58

NA

TLF

FNIL

LRA

t4g

3580

0

DN

A-d

irec

ted

RN

Ap

oly

mer

ase

IIsu

bu

nit

RP

B2

P38

420|

RP

B2_

AR

AT

H13

983.

02

16.8

0LD

LAG

PLL

GG

LFR

,LL

ICA

LGR

At4

g21

710

DN

A-d

irec

ted

RN

Ap

oly

mer

ase

IIsu

bu

nit

RP

B3-

AQ

3921

1|R

PB

3A_A

RA

TH

3543

4.0

14.

08A

SQ

LVLN

AID

LLK

At2

g15

430

DN

Ag

yras

esu

bu

nit

AQ

9CA

F6|G

YR

A_A

RA

TH

104

522.

66

8.42

ELL

QA

FID

FR,

FHP

HG

DT

AV

YD

SLV

R,

FTD

ES

SS

LTE

QIT

K,

GG

DP

ALV

LNN

LYR

,LI

EQ

EA

IELK

,N

AA

GT

PLV

QIL

SM

SE

GE

R

At3

g10

690

Oth

ers

Tu

bu

linal

ph

a-2/

alp

ha-

4ch

ain

P29

510|

TB

A2_

AR

AT

H49

556.

84

11.1

0E

IVD

LCLD

R,

FDLM

YA

K,

LVS

QV

ISS

LTA

SLR

,T

VG

GG

DD

AFN

TFF

SE

TG

AG

KA

t1g

0482

0

Tu

bu

linb

eta-

4ch

ain

P24

636|

TB

B4_

AR

AT

H49

639.

78

22.5

0A

VLM

DLE

PG

TM

DS

LR,

FPG

QLN

SD

LR,

GH

YT

EG

AE

LID

SV

LDV

VR

,LA

VN

LIP

FPR

,LH

FFM

VG

FAP

LTS

R,

NS

SY

FVE

WIP

NN

VK

,V

SE

QFT

AM

FR,

YLT

AS

AV

FR

At5

g44

340

Co

ato

mer

sub

un

itg

amm

aQ

0WW

26|C

OP

G_A

RA

TH

9860

5.0

11.

35S

IAT

LAIT

TLL

KA

t4g

3445

0E

nd

og

luca

nas

e17

O81

416|

GU

N17

_AR

AT

H24

575.

81

5.45

QV

DY

ILG

DN

PLR

At4

g02

290

NA

DP

-sp

ecifi

cg

luta

tam

ate

deh

ydro

gen

ase

Q9C

8I0|

Q9C

8I0_

AR

AT

H69

670.

91

2.08

LAG

QFQ

GS

FTG

PR

At1

g33

420

P27

SJ

(Fra

gm

ent)

Q5G

1J7|

Q5G

1J7_

HY

PP

E26

207.

21

4.56

SE

SS

GT

TE

LFT

RP

atel

lin-1

Q56

WK

6|P

AT

L1_A

RA

TH

6402

7.0

11.

75T

FGS

IITS

PR

At1

g72

150



domain-containing protein or TSN). Finally, only seven

detected proteins were not classed in any of the above

groups. The identified proteins were distributed in seven

classes, as shown in Figure 2. Approximately 79% of the

proteins identified in the BoU3 snoRNP fraction are directly

related to biogenesis and the structure and/or function of the

ribosome (Figure 2 and Discussion).

Cellular localization of BoU3 snoRNP_GFP fused proteins

In order to establish a functional connection between ribo-

some biogenesis and the identified BoU3 snoRNP proteins,

the subcellular localization of several proteins was deter-

mined. Fibrillarin/Nop1p, Nop56p, and some small and large

subunit ribosomal proteins (see Table 1) were also identified

in the proteomic analysis of nucleoli purified from Arabid-

opsis cell cultures (Pendle et al., 2005), and in some cases

the nucleolar localization was confirmed using GFP::protein

fusions expressed transiently in Arabidopsis thaliana and

tobacco (Nicotiana tabacum) cells (Barneche et al., 2001;

Pendle et al., 2005; Pontvianne et al., 2007). Our goal was to

search for novel proteins that could play a role in certain

nuclear and/or nucleolar steps of plant ribosome biogenesis.

We examined the cellular localization of HSP81, TCP, RPN1a

and eIF3H1 (Figures 3 and S3) subunits because the coun-

terparts in archaeon and yeast cells have been implicated in

pre-rRNA processing and/or ribosomal biogenesis (Ruggero

et al., 1998; Valasek et al., 2001; Schlatter et al., 2002;

Stavreva et al., 2006), but not in those from higher eukary-

otes. Moreover, we tested the cellular localization of ARCA

(Zanetti et al., 2005), a protein associated with the 40S

ribosome subunit (Figure 3). This was carried out by cloning

the cDNA sequence upstream from the enhanced green

fluorescent protein (eGFP) coding sequence to create a

chimeric sequence under the control of the 35S promoter.

The constructs were then transformed stably into BY2 cells.

Fibrillarin was used as a control for nucleolar localization

(Figure 3a–c). Observation of GFP fluorescence by micros-

copy indicates that ARCA, RPN1a and TCP-1z show nuclear

localization with a clear exclusion from the nucleolus (Fig-

ures 3d–f, j, k and S3), whereas eIF3H1 and HSP81 encode

cytoplasmic proteins (Figures 3g–i,l and S3).

Identification and analysis of plant U3 snoRNA sequences

The U3 snoRNAs co-purifying in the BoU3 snoRNP fraction

were cloned by RT-PCR. Sequence analysis of these clones

revealed three conserved and distinct U3 sequences,

named BoU3A, BoU3B and BoU3C (Figure 4). As shown in

Figure 4(b), the BoU3 sequences can be folded into a

similar triple stem-loop structure as that proposed for

Arabidopsis U3 snoRNA (Marshallsay et al., 1990). In all

sequences we identified the characteristic GAC A¢, A, B, C

and D boxes as observed in the U3 sequences from yeast,

trypanosome and vertebrates (Segault et al., 1992; Hart-

shorne and Agabian, 1994; Speckmann et al., 1999; Boro-

vjagin and Gerbi, 2001). However, significant nucleotide

variance between BoU3A, BoU3B and BoU3C is also

observed (Figure 4a,b). Nucleotide changes are observed in

the 5¢-terminal portion (hairpins 1 and 2), the hinge struc-

ture and the 3¢ terminal portion (hairpins 3 and 4).

Remarkably, we observed considerable variation in the

short stem structure located downstream from the C box

(hairpin 4). However, all the variations are structurally

compensatory and, consequently, the hairpin structure is

maintained (Figure 4b).

Phylogenetic analysis showed that the BoU3 sequences

are classified in the U3 group from dicotyledonous plants

(Figure S1). Furthermore, it is probable that U3 sequences

from Brassica and Arabidopsis diverged from a recent

duplication event in dicotyledonous plants. Moreover, anal-

ysis of U3 sequences folded using the DINAMelt Server

(Markham and Zuker, 2005) revealed a conserved two-U3

structural domain from both monocotyledonous and dicot-

yledonous plants (Figures 5 and S2). Despite a few structural

differences in the 5¢ and central regions, hairpin 4 is

maintained in the large stem loop in the 3¢ terminal region

in all predicted U3 structures (Figures 5 and S2). Another

interesting feature in cruciferous plants emerged from this

analysis. The large stem loop located in the 3¢ end region of

the B. oleracea and A. thaliana U3 sequences can be folded

into a single stem loop structure with two (BoU3A and

BoU3B) or three (BoU3C) hairpin structures. Interestingly,

the cruciform structure seems to be specific to cruciferous

DNA/RNAactivities

10%

Others11%

snoRNP/snRNP

8%

40S Ribosomalproteins

35%

Ribosomefunction

10%

60S Ribosomalproteins

8%

Chaperones/proteasome

18%

Figure 2. Distribution of the proteins identified

in the BoU3 snoRNP fraction according to the

biological process to which they are assigned: 22

small subunit ribosomal proteins (RPSs); five

large subunit ribosomal proteins (RPLs); ten

chaperones/proteasomes (one HSP 81, eight

TCP subunits and one RPN1a); five snoRNA/

snRNP (three small snoRNPs and two small

nuclear snRNPs); six ribosome function-related

proteins (ARCA and eIF3H1); six proteins related

to DNA and/or RNA metabolism (RNA polII

subunits, DNA gyrase, TSN and RdRP); and

seven unclassified polypeptides.



plants, as it is not observed in any other U3 plant sequence

analysed (Figure S2 and data not shown).

Identification of RNA fragments protected from nuclease

digestion

To determine potential RNA secondary structures and/or

sites of protein–RNA interaction in the BoU3 snoRNA, an

oligonucleotide-directed RNAse H-targeting experiment

was used (Figure 6). We found that the 3¢ end region is rel-

atively more resistant to digestion compared with the 5¢ end

of the BoU3 snoRNA sequence (Figure 6a). At the 5¢ end

(lanes 1–5), the RNA sequences that are complementary to

oligonucleotides 1–3 and 5 were particularly sensitive to

Rnase H treatment compared with sequences located just

upstream from the predicted hinge structure (lane 4). In

contrast, most of the RNA sequences in the 3¢ end region

(lanes 6–13) were resistant to different degrees to Rnase H

degradation, except for those complementary to oligonu-

cleotide 9 (lane 9). The different U3 oligonucleotides effi-

ciently targeted the U3 snoRNA sequences without affecting

the integrity of U14 snoRNA, which also purifies with the

BoU3 snoRNP fraction (lower panel, lanes 1–13). After

BoU3 snoRNP treatment with Rnase H and analysis for

rDNA binding activity, only the fraction pre-incubated with

oligonucleotide 5 (complementary to the hinge and box C¢sequences) showed a significant reduction in protein–DNA

Fibrillarin

ARCA

eIF3H1

RPN1a

*

*

TCP1z

HSP81

(a)

(d)

(g)

(j) (k) (l)

(i)(h)

(f)(e)

(c)(b)

Gene EGFP tnosP35S

Figure 3. Localization of GFP-tagged proteins in

transformed tobacco BY2 cells.

Images were observed using a Zeiss Axioscope

2. Observation was performed using Nomarski

optics (a, d, g), GFP filters (b, e, h, j–l) and merged

images (c, f, i). The arrowhead and asterisk

indicate, respectively, the nucleus and the

nucleolus. Scale bars: 20 lm.

(a–c) fusion protein Fib2::GFP used as control for

nucleolar localization.

(d–f, j, k) Fluorescence of ARCA::GFP,

RPN1a::GFP and TCP1z::GFP fusion proteins in

the nucleus, with an exclusion from the nucleo-

lus.

(g–i, l) Observation of cytoplasmic fluorescence

for the fusion proteins eIF3H1::GFP and

HSP81::GFP, respectively. The HSP81::GFP fluo-

rescence is observed as granules in the cyto-

plasm and around the nucleus (perinuclear

granules). The exact identity of these granules

remains unknown.



1 23GAC Box A’ Box A

4 67

89

1011

Hinge BxoBCxoB

Box C

1213

Box D

Hairpin 3

Hairpin 1

Hairpin 2

Hairpin 4

(a)

(b)

Figure 4. Sequences and secondary structures of BoU3 snoRNA.

(a) Sequence alignment of BoU3A, BoU3B and BoU3C clones isolated from BoU3 snoRNP. The GAC A¢, C¢, B, C and D sequences are boxed. Sequences overlined

and numbered (1–13) correspond to oligonucleotides BoU3oli1–BoU3oli13 used in the RNaseH digestion experiments (see Figure 6).

(b) The BoU3 snoRNA structure model derived from BoU3C and obtained manually based on the U3 snoRNA structure from Arabidopsis thaliana. Nucleotide

substitutions and/or insertions/deletions found in BoU3A and BoU3B sequences are indicated by arrows and are set in lower case. The GAC A¢, C¢, B, C and D

sequences are boxed, and the hinge sequences are underlined. Hairpin 4, containing major conservative substitutions, is encircled.



complex forming activity, as visualized by EMSA (Figure 6b,

lane 5). No major effect was observed in fractions pre-

incubated with oligonucleotides that affect the integrity of

BoU3, either at the 5¢ and/or the 3¢ end regions.

DISCUSSION

Here, we report on the identification of proteins and

U3 snoRNA co-purifying in the U3 snoRNP protein complex

isolated from B. oleracea. The BoU3 snoRNP complex frac-

tion was purified on the basis of its specific binding to rDNA

sequences encoding the primary cleavage site on the 5¢ ETS

of pre-rRNA.

The nanoLC-MS/MS analysis identified 62 proteins

co-purifying in the BoU3 snoRNP fraction, more than half

of which are predicted to be involved in RNA processing,

ribosome biogenesis and/or correspond to ribosomal

proteins from the small ribosome subunits (Figure 2).

Fibrillarin/Nop1, Nop5/58p, Dyskerine/Cbf5p and several

small subunit ribosomal proteins identified in this study

(Table 1) have also been found in the nucleolar proteome of

Arabidopsis (Pendle et al., 2005). Remarkably, only five

ribosomal proteins from the large ribosomal subunit (RPL)

were detected in the BoU3 snoRNP fraction, three of which

were also identified in the nucleolar proteome of Arabidop-

sis. This observation indicates either that these RPL proteins

assemble early on pre-ribosomes and/or that they can

control rRNA gene synthesis, as suggested at least for

RPL5 (Mathieu et al., 2003).

The finding of pre-RNA processing factors and RPS

proteins in the BoU3 snoRNP fraction is in agreement with

results obtained in animal and yeast systems. Indeed, yeast

and animal U3 snoRNP complexes also associate with RPS

proteins, in addition to other processing and assembly

protein factors, to form 90S pre-ribosome particles that are

generated at the end of 40S ribosomal subunits (Grandi

et al., 2002; Schafer et al., 2003; Tschochner and Hurt, 2003).

A’A

C

B

A’ AB

h4

BoU3B AtU3B

C’ DGA

C

GA

C

C’

C

D

h4

C h4

A’ A

C’

B

DGA

C

A’

GA

C

A

C’

B

CD

BoU3C AtU3D

h4

Figure 5. Predicted U3 snoRNA secondary structures from Brassica oleracea and Arabidopsis thaliana.

Predicted secondary structures of snoRNA sequences from BoU3B, BoU3C, AtU3B (AB007644.1) and AtU3D (ABO13387.1) were generated with the DINAMelt

Server. For BoU3 sequences, the hinge sequences are overlined and the GAC A¢, C¢, B, C and D boxes are indicated by brackets.



Although BoU3 snoRNP has functional and/or structural

similarities to the processome from yeast (Dragon et al.,

2002; Gallagher et al., 2004), no Utp-like proteins were

detected in the BoU3 snoRNP complex. The t-Utp complex

is a subcomplex of the yeast processome that binds rDNA

chromatin, and is required for optimal rRNA transcription

(Gallagher et al., 2004). A. thaliana, a closely related plant

species belonging to the same family as B. oleracea,

expresses at least three Utp-like proteins (Pagnussat et al.,

2005; Thiry and Lafontaine, 2005; Griffith et al., 2007). Thus,

whether or not Utp proteins interact with plant U3 snoRNP

in vivo remains an open question.

The nucleolin-like protein previously identified in BoU3

snoRNP by western blot and Edman degradation (Saez-

Vasquez et al., 2004b) was not identified during the nanoLC-

MS/MS analysis either, although NFB, a complex that is a

subcomplex of BoU3 snoRNP, contains nucleolin (Saez-

Vasquez et al., 2004b). Consequently, the most plausible

explanation for the absence of nucleolin detection is that this

protein (and eventually Utp and other proteins) might disso-

ciate from BoU3 snoRNP during purification, and/or might be

presentatconcentrationsundetectablebynanoLC-MS/MS. In

agreementwith thisobservation,a recentstudyperformedon

human cells revealed that nucleolin is present at low levels

in the human U3 snoRNP fraction, and is associated only

transiently with this complex (Turner et al., 2009).

As expected, C/D snoRNA binding proteins (fibrillarin and

Nop5/58) were detected in the BoU3 snoRNP fraction. How-

ever, it is interesting to note that diskerin/Cbf5, the pseudo-

uridine synthase activity of the H/ACA snoRNP (Meier, 2005)

and two spliceosomal proteins (SUS2/PRP8 and CLO/GFA1/

Snu114p) (Whittaker et al., 1990; Moll et al., 2008) were

identified in the BoU3 snoRNP fraction. Although the role of

diskerin/Cbf5 in rRNA maturation is well known, the role of

PRP8 and Snu114p proteins in pre-rRNA processing remains

uncertain. It is possible that plant SUS2/PRP8 and CLO/

GFA1/Snu114p proteins do not play a role in pre-rRNA

synthesis, but do play a role in U3 snoRNP biogenesis. This

is the case for some H/ACA and C/D snoRNP complexes from

yeast and vertebrates that assemble during the transcription

and splicing of intronic snoRNA (reviewed recently in Brown

et al., 2008). Although transcription of plant U3 snoRNA has

been described to be dependent on RNA polIII (Marshallsay

et al., 1990), analysis of the A. thaliana and O. sativa

genomes revealed that some U3 snoRNA sequences are

nested in protein-coding genes (data not shown). Finally, we

cannot eliminate the possibility that SUS2/PRP8 and CLO/

GFA1/Snu114 might be contaminating proteins from splic-

esomal particles. Indeed, splicesomal proteins transit

through the nucleolus during snRNA processing and snRNP

assembly (Shaw and Brown, 2004; Shaw et al., 2008).

Using a transient expression strategy we further charac-

terized some of the proteins identified in the BoU3 snoRNP.

We selected proteins with archeon and/or yeast counterparts

playing a role in early ribosome steps. We observed that

HSP81 does not localize to the nucleolus and/or nucleus as

expected for a protein involved in early pre-40S ribosome

synthesis steps (Figure 3, panel l, and Figure S3). Indeed,

HSP proteins have been associated with the processing of

the internal spacer of pre-rRNA (Lalev and Nazar, 2001) and

nuclear export of ribosome subunits (Schlatter et al., 2002).

In contrast, although excluded from the nucleolus, we

observed a clear nucleoplasmic localization for TCP1-zeta

and RPN1a (Figure 3, panels j and k, and Figure S3). In

archeon, chaperonin-like proteins interact specifically with

16S rRNA in vivo, and participate in the maturation of its 5¢extremity in vitro (Ruggero et al., 1998). More recently, it

was reported that a chaperonin from Chlamydomonas binds

group-II intron RNAs derived from mitochondrial rRNA,

suggesting a general function of this protein in RNA

2

5’ portion 3’ portion

BoU3A

BoU14

Bo

x G

AC

/A’

Hai

rpin

2

Bo

x B

Tip

Hai

rpin

3

Bo

x C

Hai

rpin

4

Bo

x D

mo

ck

Bo

x A

Hin

ge/

Bo

x C

’

Ste

m

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Bo

x G

AC

/A’

Hin

ge/

Bo

x C

’

Hai

rpin

2

Bo

x B

Tip

Hai

rpin

3

Bo

x C

Hai

rpin

4

Bo

x D

mo

ck

Bo

x A

Ste

m

T

1 2 3 4 5 6 7 8 9 10 11 12 13 14

(a)

(b)

Figure 6. Identification of U3 snoRNA fragments protected from nuclease

digestion.

The BoU3 snoRNP fraction was pre-treated with Rnase H and U3-specific

oligonucleotides (lanes 1–13), and then analysed by RT-PCR to detect U3

snoRNA and U14snoRNA (a), or were incubated with A123BP rDNA probe to

study the BoU3 snoRNP–rDNA protein interaction (b). The U3 snoRNA

sequences targeted by oligonucleotides BoU3oli1–BoU3oli13 correspond to

box GAC/A¢ (lane 1), box A (lane 2), hairpin 2 (lanes 3 and 4), hinge and box C

(lane 5), hairpin 3 (lane 6), box B (lane 7), peak of hairpin 3 (lane 8), box C (lane

10), hairpin 4 (lanes 11 and 12) and box D (lane 13). Oligonucleotide

sequences and positions are given in Figure 4(a). Lane 14, untreated BoU3

snoRNP fraction. The small black arrow shows the rDNA-BoU3 snoRNP

protein subcomplex, and the large black arrow shows the rDNA-NFB protein

complex.



metabolism (Balczun et al., 2006). Thus, the fact that chap-

eronin-like proteins co-purify in the BoU3 snoRNP indicates

that the TCP1 complex might play a major role in 5¢ ETS

pre-rRNA processing. Moreover, in agreement with the

observed nuclear localization of RPN1, it has been shown

in yeast cells that the inhibition of the proteasome affects

the accumulation of 90S pre-ribosomes, the production of

mature rRNA, and the distribution of early and late pre-rRNA

processing factors (Stavreva et al., 2006).

In Arabidopsis thaliana, mutation of the RPN1a gene

causes embryo lethality (Brukhin et al., 2005). A similar

situation was observed for other protein factors related to

ribosome biogenesis, including the plant mutants for SWA1

and TOZ genes that encode the Arabidopsis Utp15 and

Utp13 proteins (see above) that are both essential for

gametogenesis and embryogenesis (Thiry and Lafontaine,

2005; Griffith et al., 2007). The role of these factors in the

processing of 18S rRNA has been confirmed, at least for

SWA1 (reviewed in Saez-Vasquez and Medina, 2008). On the

other hand, our results describe the nuclear localization of

the ARCA protein (Figure 3, panels d–f). In the ribosome,

ARCA locates on the head of the 40S ribosomal subunit,

where it plays a major role in translation (Giavalisco et al.,

2005; Zanetti et al., 2005). The reason for its nuclear local-

ization remains unclear, but it is known that the repeated

WD40 motifs, also found in ARCA protein sequences, act as a

site for protein–protein interaction and serve as platforms

for the assembly of protein complexes (Miles et al., 2005).

The specific role of TCP1-zeta, RPN1a and ARCA proteins in

ribosome biogenesis in plants remains an open issue.

Among all the proteins identified in this study, the

detection of TSN and RNA-directed RNA polymerase (RdRP)

in the BoU3 snoRNP fraction is particularly intriguing. In

higher eukaryotic cells, the TSN protein is both an RNA and

DNA binding protein, but lacks the nucleolytic active site

residues of Staphylococcal nuclease (Abe et al., 2003).

Interestingly, the Tudor domain from the SMM protein has

been implicated in direct interaction with the GAR domain of

fibrillarin (Jones et al., 2001), suggesting that TSN and

fibrillarin from B. oleracea might interact in vivo, and may

consequently co-purify in the BoU3 snoRNP fraction. On the

other hand, it is well known that RdRPs play a key role in

RNA-mediated gene silencing (Wassenegger and Krczal,

2006). RdRP/RDR2 shows a nucleolar localization, and has

been involved in silencing of rDNA in the epigenetic

phenomenon known as nucleolar dominance (Preuss et al.,

2008). So far no interaction between RdRP and rRNA

processing factors has been reported. However, we can

reasonably hypothesize that processing of pre-rRNA and its

conversion into dsRNA, which directs rDNA silencing, might

share a number of pre-rRNA binding factors.

Another major finding of this study concerns the U3

snoRNA sequences detected in the BoU3 snoRNP fraction

(Figure 4). What is the role of hairpin 4 and U3 snoRNA

variants in the BoU3 snoRNP fraction? The hairpin 4 struc-

ture is present in the U3 sequences from yeast to humans.

Despite the fact that the role of GAC A¢, A, B, C and D boxes is

relatively well established, little is known about the structure

of hairpin 4 in these species (Segault et al., 1992; Hartshorne

and Agabian, 1994; Speckmann et al., 1999). Nevertheless, it

is possible that this hairpin contributes to the recruitment of

specific U3 snoRNA binding proteins. This is the case for an

RNA element flanking the B/C box, which is required for the

optimal binding of the hU3-55K protein (Granneman et al.,

2002). On the other hand, a biform (BoU3A and BoU3B) and

a cruciform secondary structure (BoU3C) were predicted

at the 3¢ end of the BoU3 snoRNA sequences identified

(Figure 5). Remarkably, the BoU3B cruciform structure is

also predicted for some U3 snoRNA sequences from

A. thaliana, but not in other monocotyledons and/or dicoty-

ledons (Figure S2). Thus, the cruciform structure seems to

be specific to ‘cruciferous plants’. Although the functional

significance of these structures in vivo remains unclear,

nuclease protection experiments (Figure 6) indicate that

the 3¢ region of BoU3 is highly structured and/or protected

from digestion by RNA binding factors co-purifying in the

BoU3 snoRNP fraction, in contrast with the 5¢ region.

In conclusion, structural analysis of the functional BoU3

snoRNP complex allowed us to identify novel proteins and

RNA structures that are potentially involved in rRNA synthe-

sis and/or other ribosome assembly steps in plants. Some

of the identified factors are certainly contaminant proteins

co-eluting throughout the biochemical steps of purification,

but it is also clear that many of the identified nuclear proteins

(TCP1-zeta, RPN1a and ARCA) could be related in one way

or another to ribosome biogenesis. Finally, it is clear that

this analysis revealed unexpected protein factors (TSN and

RdrRP) and RNA structures that might play a major role in

RNA synthesis and RNP complex assembly, not only in

plants but also in other higher eukaryotic organisms.

EXPERIMENTAL PROCEDURES

Purification of BoU3 snoRNP

Cauliflower protein extracts precipitated with ammonium sulphatewere fractionated using DEAE-sepharose CL-6B, Heparin sepharoseand oliA DNA sepharose, as described previously (Saez-Vasquezet al., 2004b), and as shown in Figure 1a. The heparin F600 fractionwas dialysed against buffer II (50 mM Tris–HCl, pH 8, 6 mM MgCl2,15% glycerol, 1 mM EDTA and 2% NP40) containing 100 mM KCl andsubjected to oliA DNA sepharose equilibrated with buffer II-100. Thecolumn was washed with buffer II-100 and proteins were eluted withbuffer II-350, and then with buffer II-1000. Fractions were dialysedagainst buffer II-100 and were stored at )80�C. The numbers afterbuffer II and/or fraction F indicate the concentration of KCl to be inthe mM range.

DNA binding activity

For DNA binding assays, the A123BP fragment was 5¢-end labelledusing Klenow fragment and [a-32P]dCTP. Between 5 and 10 fmol of



gel-purified A123BP fragment was mixed with 15 ll of the purifiedprotein samples. After incubation of the reaction mixtures for20 min on ice, binding products were analysed by electrophoresismobility shift assay (EMSA), as described previously (Saez-Vasquezet al., 2004b).

Rnase H digestion

RNase H treatment was performed according to Kass et al. (1990).Briefly, 15 ll of the purified BoU3 snoRNP fraction were mixed with1 ll (100 lM) of oligonucleotide BoU3Oli1-13 (Appendix S2) and1 ll (5 U ml)1) Rnase H (USB, http://www.usbweb.com). Afterincubation (10 min at 37�C and 20 min at 30�C), the reaction mix-tures were treated with 1 ll (2.2 U ll)1) of Dnase I (Worthington,http://www.worthington-biochem.com) for 3 min at 37�C, followedby 7 min at 30�C. The remaining U3 snoRNAs were analyzed using1 ll of the reaction mix and by performing a one-step RT-PCR with aplatinum Taq system (Invitrogen, http://www.invitrogen.com). Toverify that oligonucleotides BoU3Oli1–BoU3Oli13 specifically directthe cleavage of U3 snoRNA, the integrity of U14 snoRNA treatmentwas controlled using oligonucleotides 5¢-AtU14 and 3¢-AtU14. Totest the BoU3 snoRNP binding activity after the total and/or partialremoval of U3 snoRNA, the S300 peak fraction (Saez-Vasquez et al.,2004b) was mixed with oligonucleotides BoU3Oli1–BoU3Oli13 andRnase H. After 10 min at 37�C and 20 min at 30�C, the reactionmixtures were incubated with the 5¢-end-labelled A123BP fragmentand then analysed by EMSA.

Analysis of U3 snoRNA sequences

BoU3A, BoU3B and BoU3C from the BoU3 snoRNP fractionwere cloned by RT-PCR and then sequenced on a ABI3100 DNAsequencer using an ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems, http://www.appliedbiosystems.com). The other plant U3 snoRNA se-quences (Appendix S1) were obtained from the RNA functionaldatabase (http://www.ncrna.org/frnadb). The phylogenetic tree forU3 snoRNA was generated by CLUSTALW 2.0.10 (Larkin et al., 2007)using the neighbour-joining method coupled with 1000 bootstraptests. U3 snoRNA secondary structures were predicted using theDINAMelt Server DINAMelt web server for nucleic acid meltingprediction (Markham and Zuker, 2005).

Cloning and cellular localization of GFP fusion proteins

Complete cDNA sequences for TCP1 (RAFL_08-18-F20), HSP81(RAFL_09-06-O18), RPN1a (RAFL_09-17-J10), ARCA (RAFL_04-13-D02) and eIF3H1 (RAFL_05-13-M08) were obtained from the RIKENBioresource Center (Yokohama, Japan). Gateway cassettes witheach coding sequence were generated by PCR following Invitro-gen’s instructions. PCR products were introduced first intopDONR207 and then cloned by recombination into pK7FWG2 toproduce TCP1::GFP, HSP81::GFP, RPN1a::GFP, ARCA::GFP,eIF3H1::GFP and FIB2::GFP plasmids. Constructs were introducedinto the Agrobacterium tumefaciens LBA4404 strain, and were usedto transform tobacco BY2 cells as described previously (Chabouteet al., 2000). Observations were performed using Nomarski opticsand a GFP filter with a Zeiss Axioscope 2 microscope (Zeiss, http://www.zeiss.com). Images were taken with a Leica DC350FX camera(Leica, http://www.leica.com).

In-gel digestion and mass spectrometry analysis

In-gel digestion and mass spectrometry analyses were performedon a CapLC (Waters, http://www.waters.com) coupled to a hybrid

quadrupole orthogonal acceleration time-of-flight tandem massspectrometer (Q-TOF 2; Waters), as described in Appendix S3.

Protein identification

The MS and MS/MS data were analysed using a local Mascot server(MASCOT 2.0; MatrixScience, http://www.matrixscience.com) bycomparison with a composite target–decoy database, including theUniProt protein sequences of Viridiplantae (554 832 sequences;January 2009), human keratins, porcine Trypsin and all corre-sponding reversed sequences (1 109 972 entries in total).

Searches were performed with a mass tolerance of 250 ppm inMS mode and 0.4 Da in MS/MS mode for nanoLC-MS/MS data. Onemissed cleavage per peptide was allowed and variable modifica-tions were taken into account, such as carbamidomethylation ofcysteine, oxidation of methionine and N-acetylation (protein N-ter).Neither protein molecular weight nor isoelectric point constrainswere applied (see Figure S4).

Mascot results were loaded in MuDPIT mode into SCAFFOLD

(Proteome Software, http://www.proteomesoftware.com). Resultswere subjected to the following filtering criteria. For the identifica-tion of proteins with two peptides or more, a Mascot ion score ofabove 25 was required. In the case of single peptide hits, the score ofthe unique peptide must be greater (minimal ‘difference score’ of 0)than the 95% Mascot significance threshold. The spectra of thosesingle peptide hits are provided in Figure S5). The target–decoydatabase search allows us to control and estimate the false-positiveidentification rate of our study (Peng et al., 2003; Elias and Gygi,2007). Thus, the final catalogue of proteins presents an estimatedfalse-positive rate of below 1%.

ACKNOWLEDGEMENTS

This work was supported by the Centre National de la RechercheScientifique. FP and HS were supported by fellowships from theMinistere de l’Enseignement et de la Recherche and the Lebanesegovernment, respectively. The authors also thank C. Chaparro forhelp in obtaining U3 snoRNA sequences and in the generation ofthe phylogenetic tree.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Phylogenetic relationships of different plant U3 snoRNAsequences.Figure S2. Predicted secondary structures of plant snoRNAsequences.Figure S3. Localization of GFP-tagged RPN1a TCP1z and HSP81proteins.Figure S4. Distribution of the errors measured on the precursor ionmasses.Figure S5. MS/MS spectra of single peptide hits.Table S1. Complete list of proteins identified in the U3 snoRNPcomplex isolated from Brassica oleracea.Appendix S1. Sequences used for phylogenetic and structuralanalysis.Appendix S2. Oligonucleotides used in this work.Appendix S3. Mass spectrometry analysis and protein identification.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.



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