Arabidopsis CBF5 interacts with the H/ACA snoRNP assemblyfactor NAF1

Inna Lermontova Æ Veit Schubert Æ Frederik Bornke ÆJiri Macas Æ Ingo Schubert

Received: 9 May 2007 / Accepted: 9 August 2007 / Published online: 22 August 2007

� Springer Science+Business Media B.V. 2007

Abstract The conserved protein CBF5, initially regarded

as a centromere binding protein in yeast and higher plants,

was later found within nucleoli and in Cajal bodies of yeast

and metazoa. There, it is assumed to be involved in post-

transcriptional pseudouridinylation of various RNA species

that might be important for RNA processing. We found

EYFP-labeled CBF5 of A. thaliana to be located within

nucleoli and Cajal bodies, but neither at centromeres nor

somewhere else on chromosomes. Arabidopsis mutants

carrying a homozygous T-DNA insertion at the CBF5 locus

were lethal. Yeast two-hybrid and mRNA expression

analyses demonstrated that AtCBF5 is co-expressed and

interacts with a previously uncharacterized protein con-

taining a conserved NAF1 domain, presumably involved in

H/ACA box snoRNP biogenesis. The homologous yeast

protein has been shown to contribute to RNA pseudou-

ridinylation. Thus, AtCBF5 might have an essential

function in RNA processing rather than being a kineto-

chore protein.

Keywords Cajal bodies � Centromere � H/ACA snoRNP �Nucleolus � Pseudouridine synthase

Abbreviations

3AT 3-amino-triazole

BiFC Bimolecular fluorescence

complementation

CB Cajal bodies

CDD Conserved domain database

DAPI 40,6-diamidino-2-phenylindole

DIC Differential interference contrast

PPT Phosphinotricine

snRNAs Small nuclear RNAs

snRNPs Small nuclear ribonucleoproteins

snoRNAs Small nucleolar RNAs

snoRNPs Small nucleolar ribonucleoproteins

TMG-capped-

snRNA

tri-methylguanosine-capped

snRNA

W Pseudouridine

Introduction

The Cbf5p protein of Saccharomyces cerevisiae (ScCBF5)

was initially isolated as a low-affinity centromere DNA

binding protein (Jiang et al. 1993). The CBF5 protein is

highly conserved among different phyla. ScCBF5 is 71%

identical to NAP57, a related protein of mammals

(mCBF5; Meier and Blobel 1994), and 70% to Nop60B of

Drosophila (DmCBF5; Philips et al. 1998). Neither the

centromeric location nor a centromere function were

observed for Drosophila (Philips et al. 1998), rat (Meier

and Blobel 1994) or human (Kukalev et al. 2005). In

contrast, antibodies directed against a peptide derived from

a putative CBF5 homologue of barley labeled preferen-

tially the centromeres of isolated metaphase chromosomes

of barley and Vicia faba, respectively (tenHoopen et al.

2000).

I. Lermontova (&) � V. Schubert � I. Schubert

Leibniz Institute of Plant Genetics and Crop Plant Research

(IPK), 06466 Gatersleben, Germany

e-mail: lermonto@ipk-gatersleben.de

F. Bornke

Department of Biochemistry, Friedrich-Alexander University

Erlangen-Nurnberg, Straudtstrasse 5, 91058 Erlangen, Germany

J. Macas

Institute of Plant Molecular Biology, Branisovska 31, 37005

Ceske Budejovice, Czech Republic

123

Plant Mol Biol (2007) 65:615–626

DOI 10.1007/s11103-007-9226-z

In yeast, Drosophila and mammals, CBF5 is located

within nucleoli (Cadwell et al. 1997; Philips et al. 1998;

Meier and Blobel 1994). Immunostaining and immuno-

electron microscopy experiments localized rat NAP57

(RnCBF5) additionally at extranucleolar dots which were

identified as ‘coiled’ or Cajal bodies (Meier and Blobel

1994).

The nucleolar and Cajal body (CB) location of CBF5

might suggest a function unrelated to centromeres and

chromosome segregation, since the main role of the

nucleolus is the transcription, maturation and packaging of

rRNA into ribosomal particles (Melese and Xue 1995;

Shaw and Jordan 1995). CBs store spliceosomal proteins,

small nuclear and nucleolar ribonucleoproteins (snRNPs

and snoRNPs), components of the basal transcription, as

well as components of the RNAi machinery and are often

associated with the nucleolus (Andrade et al. 1993; Brasch

and Ochs 1992; Darzacq et al. 2002; Li and Ye 2006).

ScCBF5 and RnCBF5 are distantly related to the TruB

tRNA pseudouridine 55 synthase of E. coli (Meier and

Blobel 1994; Koonin 1996), which catalyzes the isomeri-

sation of uridine into pseudouridine (W) at position 55 of

prokaryotic tRNA (Nurse et al. 1995). W is the most

abundant nucleoside modification in tRNA and rRNA and

occurs in small nuclear RNAs (snRNAs) and in small

nucleolar RNAs (snoRNAs) (Gu et al. 1998; Massenet

et al. 1998). Despite the abundance of W in various classes

of RNA, little is known about its biological importance. Wbases may serve to stabilize the secondary structure of

tRNA (Arnez and Steitz 1994). In snRNAs, W bases are

required for snRNP function in splicing (Zhao and Yu

2007). The nucleolar localization of ScCBF5 and mCBF5

and their similarity to bacterial pseudouridine synthase

suggested that CBF5 may catalyze W formation (Koonin

1996), the more so as depletion of ScCBF5 prevents

pseudouridinylation of pre-rRNA (Lafontaine et al. 1998).

Most of the RNA modifications are guided by site-specific

base pairing with snoRNAs (Bachellerie et al. 2002; Ganot

et al. 1997; Kiss-Laszlo et al. 1996; Ni et al. 1997). The

snoRNAs that guide pseudouridinylation possess a specific

sequence motive, the H/ACA box (Kiss et al. 2002; Tol-

lervey and Kiss 1997), and constitute parts of specific

snoRNP particles. So far, four H/ACA box proteins

(GAR1, CBF5, NHP2 and NOP10) were identified (Kiss

2001).

The removal of intron sequences of mRNA precursors is

catalyzed by spliceosomes, a dynamic assembly of five

snRNAs and numerous protein factors (Darzacq et al.

2002). The spliceosomal snRNAs contain many posttran-

scriptionally inserted W (Reddy and Busch 1983; Massenet

et al. 1998). Pseudouridinylation of snRNA takes place in

CBs and regards regions known to interact with pre-

mRNAs, other snRNAs or spliceosomal proteins (Darzacq

et al. 2002). CBF5 seems to be involved in pseudourid-

inylation of snRNAs, since depletion of the Trypanosoma

brucei pseudouridine synthase, TbCBF5, a H/ACA box

snoRNP protein, yielded snRNAs with reduced amounts of

W (Barth et al. 2005).

In this study, a functional analysis of CBF5 in Arabid-

opsis is described. Subcellular localization studies on

transgenic Arabidopsis plants that express a CBF5-EYFP

fusion construct revealed localization of the fusion protein

within nucleoli and sometimes in additional nuclear bodies

at the periphery or outside the nucleoli. During mitosis no

fluorescent signals were detected on chromosomes. Im-

munostaining of meristematic nuclei of transformants with

anti-GFP and with anti-tri-methylguanosine-capped

snRNA (TMG-capped-snRNA) antibodies revealed colo-

calization of signal aggregations in nuclear bodies, often

associated with the nucleolus. In differentiated nuclei of

different ploidy level, CBF5 and snRNAs both occupied

the entire nucleolus as well as nuclear bodies apparently

extruding from nucleoli. Yeast two-hybrid screening of an

A. thaliana cDNA library using AtCBF5 as a bait identified

a previously uncharacterized protein with similarity to the

yeast NAF1 protein required for the stability of H/ACA

box snoRNPs (Yang et al. 2002) as a potential interaction

partner of AtCBF5. Bimolecular fluorescence comple-

mentation (BiFC) experiments in Nicotiana benthamiana

confirmed an interaction of AtCBF5 and AtNAF1 in

planta.

Materials and methods

Gene cloning

Pooled mRNA of seedling, flower bud, root, leaf and stem

RNA was isolated using magnetic beads (Dyanal, Oslo,

Norway). The full-length cDNA of A. thaliana CBF5 was

amplified with the primer pair 50- GAC ACT AGT ATG

GCG GAG GTC GAC ATC TCA C -30; and 50- TCC GAA

TTC TTC CTC ATC ATC CTC ACT GTC T -30 by RT-

PCR (RevertAid HMinus First Strand cDNA Synthesis kit;

Fermentas, St. Leon- Rot, Germany). At the 50-end of the

primers, a restriction recognition site specific for SpeI and

EcoRI, respectively, was added. The RT-PCR product was

cloned in the pCR 2.1-TOPO (Invitrogen, Carlsbad, Cali-

fornia) vector to generate the pCR2.1-TOPO/CBF5

plasmid.

Generation of CBF5-EYFP fusion construct

The EYFP DNA sequence was amplified with the primer

pair 50- AGC AAG CTT ATG GTG AGC AAG GGC GAG

616 Plant Mol Biol (2007) 65:615–626

123

GAG -30 and 50-TCT GTC GAC TTA CTT GTA CAG

CTC GTC CAT G -30 from the pWEN18 vector, generating

a HindIII linker sequence at the 50 end and a SalI linker

sequence at the 30 end. The amplified fragment was

inserted downstream the 35S promoter in the unique

HindIII and SalI sites of the p35S-BAM vector

(http://www.dna-cloning-service.de). To generate the

p35S:CBF5-EYFP fusion construct, the CBF5 sequence

was cut out from pCR2.1-TOPO vector using SpeI and

EcoRI, and inserted in frame with EYFP into the p35S-

BAM-EYFP vector digested with the same restriction

enzymes. The resulting expression cassette including 35S

promoter, CBF5-EYFP and Nos terminator was subcloned

into the pLH7000 vector containing the phosphinotricine

(PPT)-resistance marker (http://www.dna-cloning-service.de)

via the SfiI restriction site.

Plant transformation and analysis of EYFP expression

in vivo

Plants of A. thaliana accession Col were transformed

according to the flower-dip method (Bechtold et al. 1993).

After surface sterilization of seeds, transgenic CBF5-

EYFP-containing progeny was selected on MS medium

(Murashige and Skoog 1962) containing 16 mg/l PPT.

Growth conditions in a cultivation room were 20�C 16 h

light/18�C 8 h dark. V. faba hairy roots transgenic for

CBF5-EYFP were obtained as described (Lermontova et

al. 2006). For in vivo EYFP analysis in A. thaliana, 7- to

10-day-old seedlings were counterstained with 40,6-diami-

dino-2-phenylindole (DAPI, 1 lg/ml), placed on slides in a

drop of water and inspected using a Zeiss epifluorescence

microscope (Axiophot) equipped with 25·/0.80, 63·/1.4

and 100·/1.3 plan apochromat objectives and a 3-chip

CCD Sony color camera (DXC-950P). The microscope was

integrated into a Digital Optical 3D Microscope system

(Schwertner GbR, Germany) to generate 3D extended

focus images. For time-lapse microscopy, seedlings of

transformants were growing in coverslip chambers (Nalge

Nunc International, USA) for 7–10 days and analyzed

using a Zeiss inverted fluorescence microscope Axiovert

100TV equipped with a 63·/1.4 plan apochromat objective

and a black and white camera (CV-M300, JAE Corpora-

tion, Japan) or using a confocal laser-scanning-microscope

LSM 510 META (Zeiss, Germany) with a laser of 488 nm.

Preparation and immunostaining of chromosomes

and nuclei

For in situ detection of CBF5-EYFP and TMG-capped

snRNA, seeds of wild type (accession Columbia) and

transformed A. thaliana plants were germinated in Petri

dishes on wet filter paper for 3 days at room temperature.

Slides for immunostaining were prepared as described

(Lermontova et al. 2006). Slides for immunostaining of

N. benthamiana leaf nuclei after infiltration with BiFC

constructs were prepared as described for A. thaliana

except that digestion with the PCP enzyme mixture (2.5%

pectinase, 2.5% cellulase ‘Onozuka R-10’ and 2.5% Pec-

tolyase Y-23) dissolved in MTSB (50 mM PIPES, 5 mM

MgSO4, and 5 mM EGTA, pH 6.9) was extended from 10

to 30 min. To detect CBF5-EYFP in isolated nuclei from

roots and leaves, seeds of transgenic A. thaliana plants

were germinated in Petri dishes on wet filter paper for

5–6 days at room temperature. For isolation and flow-

sorting of nuclei using a FACSAria (BD Biosciences) see

Lermontova et al. (2006). CBF5-EYFP was detected with

rabbit polyclonal antisera against GFP (1:500, BD Bio-

sciences) and goat anti-rabbit rhodamine (1:100, Jackson

Immuno Research Laboratories). TMG-capped snRNA was

detected with mouse monoclonal antibodies (1:200, Cal-

biochem) and goat anti-mouse Alexa 488 (1:200,

Molecular Probes). CBF5-NYFP was detected with anti-c-

Myc (1:200, Medical and Biological Laboratories) and

NAF1-CYFP with anti-HA (1:200, Medical and Biological

Laboratories) antibodies and goat anti-rabbit rhodamine as

secondary antibodies (1:100, Jackson Immuno Research

Laboratories). Immunostaining of nuclei and chromosomes

was as described (Jasencakova et al. 2000).

Yeast two-hybrid screening of an A. thaliana cDNA

library

Yeast two-hybrid techniques were performed according to

the yeast protocols handbook and the Matchmaker GAL4

Two-hybrid System 3 manual (Clontech) using the yeast

reporter strain AH109. Full-length cDNA of CBF5 was

amplified by PCR from the plasmid pCR2.1-TOPO/CBF5

with the primer pair 50-ATA GAA TTC ATG GCG GAG

GTC GAC ATC TCA C-30 and 50- TAC GGA TCC TCA

TTC CTC ATC ATC CTC ACT G -30 containing EcoRI

and BamHI restriction sites, respectively. The resulting

DNA fragment was purified, cleaved with EcoRI and

BamHI and inserted into EcoRI/BamHI cleaved pGBKT7

vector (Clontech) generating a fusion with the GAL4

DNA-binding domain (BD). Using the pGBKT7-CBF5 as a

bait, we screened 2 · 105 prey clones of a two-hybrid

library form Arabidopsis inflorescence (Fan et al. 1997;

kindly provided by the Arabidopsis Biological Resource

Center) as fusions with the GAL-4 activation domain (AD).

Cells were selected on medium lacking leucine (Leu–),

tryptophane (Trp–) and histidine (His–) supplemented with

4 mM 3-amino-triazole (3AT). Cells growing on selective

Plant Mol Biol (2007) 65:615–626 617

123

medium were further tested for activity of the lacZ reporter

gene using filter lift assays. Plasmids from eight randomly

chosen his3/lacZ positive clones were isolated from yeast

cells and transformed into E. coli by electroporation before

sequencing of the cDNA inserts.

Bimolecular fluorescence complementation (BiFC)

The binary BiFC plant transformation vectors pSPYNE-

35S and pSPYCE-35S, containing a c-Myc or HA affinity

tag, respectively, were kindly provided by Klaus Harter

(University of Tubingen, Germany). The entire coding

regions of CBF5 and NAF1 were cloned into each of the

BiFC vectors in frame with the split YFP. BiFC was per-

formed in 6-week-old N. benthamiana plants after

Agrobacterium-mediated transient transformation accord-

ing to Walter et al. (2004).

Identification and analysis of T-DNA insertion mutants

The atcbf5-1 (SAIL-1149H09) and atcbf5-2 (SAIL-

697C07) mutant alleles were obtained from the Syngenta

collection of T-DNA insertion mutants (Sessions et al.

2002). Segregation of the PPT resistance marker encoded

by T-DNA tags was assayed by growing seedlings in MS

medium containing 16 mg/l PPT. To confirm the presence

of T-DNA insertions we performed PCR with pairs of

gene-specific CBF5-LP(50-CGCCTCACAAGAAACCCA-

GAA-30), CBF5-RP(50-TGGACCTCGTTTATTTGGGCA-

30) primers and with the T-DNA end-specific primer LB3

(50-TAGCATCTGAATTTCATAACCAATCTCGATACA

C-30). The gene-specific primers were used in subsequent

PCRs to classify the heterozygous and homozygous mutant

lines. PCR-amplified T-DNA-plant DNA junction frag-

ments were sequenced.

Results

CBF5-EYFP is localized within nucleoli and

subnuclear bodies but is not detectable at centromeres

In the A. thaliana genome, there is a CBF5 gene

(At3g57150) corresponding to the previously identified

AtNAP57 (Maceluch et al. 2001). Since BLAST searches

revealed no other CBF5-like sequences in the database, it

was suggested to be a single-copy gene. To study the

subcellular localization of CBF5 in plants, a CBF5-EYFP

fusion construct was expressed in A. thaliana under the

control of CaMV 35S promoter. CBF5-EYFP fluorescence

was detected preferentially in nucleoli of living leaf and

root tip cells (Fig. 1A–C). In some cases distinct signals

were also observed in nucleoplasm, clustered around the

nucleoli. Extra-nucleolar signals never coincided with the

bright DAPI stained chromocenters (Fig. 1D). During

mitosis (prophase to telophase) no CBF5-EYFP signals

were detected on the chromosomes and only weak fluo-

rescence appeared in mitotic cells (Figs. 1A, 2A).

Because the absence of fluorescent signals on chromo-

somes might be due to only a small number of fusion

protein molecules at centromeres of the relatively small

A. thaliana chromosomes, immunostaining with anti-GFP

antibodies was performed on nuclei and chromosomes of

CBF5-EYFP transformants. In meristematic nuclei, im-

munosignals of CBF5-EYFP were detected mainly at the

periphery of nucleoli and in nuclear bodies attached to

nucleoli (Fig. 1E). In some cases, additionally to the

strongly fluorescent nucleolar periphery and nuclear bod-

ies, weaker immunofluorescence was equally distributed

within nucleoplasm (Fig. 1F). Again no signals were

observed at centromeres in neither of the cell cycle stages.

In isolated differentiated nuclei of a DNA content corre-

sponding 2C, 4C, 8C and 16C, respectively, the CBF5-

EYFP signals were homogeneously distributed within the

nucleolus and additionally aggregated in bodies associated

with the nucleolus (Fig. 1G). Also, like in meristematic

cells, weaker and homogenous fluorescence was in some

nuclei distributed over the nucleoplasm (data not shown).

In rare cases, differentiated nuclei displayed only clustered

signals dispersed within the nucleoplasm (Fig. 1H).

Time-lapse microscopy of root tip cells from stably

expressing transgenic A. thaliana lines revealed CBF5-

EYFP within nucleoli during the entire interphase (Fig. 2

A). The transition from G2 until cytokinesis takes about

20 min and only weak dispersed fluorescence remained

within cytoplasm after break down of the nuclear envelope

and disappearance of nucleoli. During cytokinesis CBF5

rapidly accumulates again within the nucleoli of the

daughter cells. Interphase root tip nuclei of CBF5-EYFP

expressing A. thaliana were observed over 4.5 h under the

inverted fluorescence microscope; images were taken

within 30 min intervals. The nuclear bodies within nuclei

harbouring large nucleoli changed slowly their positions.

After *3 h no nuclear bodies were observed any more and

nucleoli decreased significantly in size during the next

1.5 h (Fig. 2B). This nuclear appearance is characteristic

for the transition from the meristematic state with a large

nucleolus, into a differentiated state with a smaller

nucleolus in the root elongation zone.

CBF5-EYFP transformed hairy roots of V. faba were

generated with the aim to check the distribution of CBF5-

EYFP in nuclei of a species with large chromosomes.

Again, the fusion protein showed similar localization as in

A. thaliana (Fig. 1I) and no labeling was detected at

618 Plant Mol Biol (2007) 65:615–626

123

centromeres of mitotic chromosomes; instead, the chro-

mosomes were embedded in cytoplasm displaying CBF5-

EYFP fluorescence uniformly (Fig. 1J).

CBF5 colocalizes with TMG-capped snRNA, a marker

for Cajal bodies

Pseudouridinylation of rRNA within the nucleolus and of

snRNA within CBs is apparently mediated by CBF5 and

the H/ACA box snoRNAs. Since also CBF5-EYFP fluo-

rescent signals were localized within nucleoli and

subnuclear bodies, we performed a double immunostaining

with anti-GFP and with antibodies against tri-methylgu-

anosine-capped snRNAs, a marker for CBs, and observed

colocalization of CBF5-EYFP and TMG-capped snRNAs

in meristematic and differentiated nuclei. In meristematic

nuclei, CBF5 and TMG-capped snRNA colocalization

regarded protein bodies attached to nucleoli (Fig. 3A),

while CBF5 was additionally observed at the periphery of

nucleoli, and TMG-capped snRNA within the remaining

nucleoplasm. In differentiated cells, colocalization applied

to nucleoli as well as to nuclear bodies (in most cases one

per nucleus) associated with or extruded from nucleoli

(Fig. 3B, C). No obvious difference as to the distribution of

CBF5 and TMG-capped snRNA were observed between

nuclei of different ploidy (Fig. 3B-C).

CBF5 interacts with a protein containing the conserved

NAF1 domain

Since the localization of CBF5 in A. thaliana nuclei did not

indicate a contribution to kinetochore formation, we

screened an A. thaliana cDNA library with the yeast two-

hybrid system to find interaction partners potentially

involved in centromere function. Screening of about

2 · 105 independent clones identified 70 colonies showing

activity of both reporter genes and thus expressing poten-

tial CBF5 interaction partners. Sequencing the cDNA

inserts of eight randomly chosen potential CBF5 interac-

tion partners and subsequent database searches revealed

that they all matched to At1g03530, a previously unchar-

acterized protein of 801aa and a predicted molecular

weight of 88.8 kD. The specificity of the CBF5-At1g03530

interaction in yeast was confirmed by retransformation of

the respective plasmids into the yeast reporter strain and

subsequent analysis of reporter gene activation (data not

Fig. 1 Localization of CBF5-EYFP in nuclei of transgenic A.thaliana and V. faba. (A) CBF5-EYFP signals (green) in root tip

meristem cells of transgenic A. thaliana, (B) CBF5-EYFP signals

(green) and DAPI staining of nuclei and chromosomes (blue), (C) the

same region as in (A) and (B) as a differential interference contrast

(DIC) image. (D) Spots of nucleoplasmic CBF5-EYFP signals (green)

do not coincide with bright DAPI stained chromocenters (blue). (E–

H) Immunostaining with anti-GFP antibodies (red) of meristematic

and differentiated nuclei of A. thaliana CBF5-EYFP transformants:

(E) localization of CBF5-EYFP at the nucleolar periphery and in

nuclear bodies, and (F) at the nucleolar periphery, in a nuclear body

and over nucleoplasm of meristematic nuclei, (G) CBF5-EYFP

immunosignals within the entire nucleolus, and (H) rare clustering of

signals within nucleoplasm of differentiated nuclei. (I) In vivo

fluorescence of CBF5-EYFP (green) within nucleolus, nuclear bodies

and nucleoplasm of a transformed V. faba hairy root cell, (J) CBF5-

EYFP signals surround DAPI-stained mitotic chromosomes (blue) of

a transformed V. faba hairy root cell. Arrows, nucleoli; arrow heads,

nuclear bodies. Bars = 20 lm for (A–C), 2 lm for (D–H) and 10 lm

for (I–J)

Plant Mol Biol (2007) 65:615–626 619

123

Fig. 2 Time-lapse microscopy

of A. thaliana root tip cells

stably expressing CBF5-EYFP.

(A) CBF5-EYFP fluorescence

(yellow) monitored during

mitotic division of two root tip

cells (1 and 2) in 10 min

intervals combined with DIC

(right). (B) A. thaliana root tip

nucleus observed under an

inverted fluorescence

microscope in 30 min intervals:

the nuclear body (arrow head)

changed slowly its position and

was no longer visible after 3 h;

after 4.5 h the nucleolus had

decreased its size as typical for

the transition from the

meristematic to the

differentiated state.

Bar = 10 lm for (A) and 2 lm

for (B)

620 Plant Mol Biol (2007) 65:615–626

123

shown). BLAST analysis of the At1g03530 protein

sequence at NCBI (http://www.ncbi.nlm.nih.gov/) via

conserved domain database (CDD) (Marchler-Bauer et al.

2005), revealed a conserved RNA binding domain NAF1

ranging from aa349 to aa512 (Fig. 4A–C). The detailed

analysis of the protein sequence revealed the following

features: a Glu/Ser/Asp-rich domain from aa233 to aa347

and a C-terminal Gln-rich (Q-rich) domain (aa581–769)

(Fig. 4A). The Q-rich domain might be important for the

interaction of NAF1 with snRNP proteins, since Q-rich

domains have often been involved in protein–protein

interactions (McBride and Silver 2001) and, moreover,

Forch et al. (2002) showed that C-terminal Q-rich domain

of the splicing regulator TIA-1 is required for association

with U1 snRNP. Originally, the NAF1 protein of yeast was

found to be required for stability of H/ACA box snoRNA

and to interact with CBF5 (Yang et al. 2002). Moreover,

according to CDD, the AtNAF1 domain is similar to the

RNA binding domain GAR1 of known GAR1 proteins and

also of the putative GAR1 protein family (GAR1-1,

At3g03920 and GAR1-2, At5g18180) of A. thaliana

(Fig. 4B). The GAR1 protein is known as part of the H/

ACA box snoRNP complex and as an interacting partner of

CBF5 (Wang and Meier 2004; Darzacq et al. 2006). Thus,

our observation corroborates the assumption that AtCBF5

might be involved in modification of RNAs. In contrast to

CBF5, which is highly conserved among different organ-

isms, NAF1 domain containing proteins are more variable.

AtNAF1 is only 38% identical with its rice homologue in a

region corresponding aa327 to 778 of A. thaliana and

aa216 to 659 of the O. sativa protein and 61% identical

within the NAF1 domain. Neither the N-(aa1–348) nor the

C-terminal (aa513–801) parts of At1g03530 protein share

any significant similarity to any other known proteins

(including NAF1 proteins from different organisms)

deposited in the public domain. Most conserved between

NAF1 proteins of different organisms is the NAF1 domain

itself (Fig. 4C). Analysis of A. thaliana co-response data-

base (http://csbdb.mpimp-golm.mpg.de, Steinhauser et al.

2004) revealed similar transcription patterns for CBF5 and

NAF1 genes in a multi-conditional set of expression

experiments. Sperman’s rank order correlation coefficient

(rs, Rautengarten et al. 2005) which ranges from +1 to –1

(the closer the correlation is to either +1 or –1, the stronger

the relationship) for CBF5 and NAF1 is 0.729. Moreover,

expression of putative GAR1, NOP10, NHP2 genes

encoding protein components of the H/ACA box snoRNP

complex, and a number of other genes involved in RNA

modification was also found to be correlated with that of

CBF5 and NAF1 (Table 1).

CBF5 and NAF1 interact in vivo

To verify interaction of CBF5 and NAF1 in planta, BiFC

assays were performed in Agrobacterium-infiltrated

tobacco (Nicotiana benthamiana) leaves (Walter et al,

2004). When CBF5 fused to the N- and NAF1 to the C-

terminal YFP fragment and vice versa were coexpressed in

tobacco leaves, BiFC signals were detected in the nuclei of

the transformed cells in case of both combinations

(Fig. 5A), confirming the interaction of AtCBF5 and

Fig. 3 Colocalization of CBF5-

EYFP and TMG-snRNA in

meristematic and differentiated

nuclei of A. thaliana after

immunostaining with anti-GFP

(red) and anti-TMG-snRNA

(green) antibodies. (A)

Immunostaining on nuclei of

squashed root tip meristem, (B)

on sorted 4C and (C) 8C nuclei.

Arrows, nucleoli; arrow heads,

Cajal bodies. Bars = 2 lm

Plant Mol Biol (2007) 65:615–626 621

123

AtNAF1 in planta. More detailed analysis of BiFC signals

in tobacco cells revealed three different localization pat-

terns (Fig. 5B–D). In most cases signals were localized in

nucleoplasm (Fig. 5B). In some cases fluorescence was

detected within nucleoplasm and nuclear bodies (Fig. 5C)

and in few cases within nucleoplasm, nuclear bodies and

Fig. 4 Schemes and sequence

comparison of NAF1 protein.

(A) Schematic view of the A.thaliana and S. cerevisiae NAF1

proteins. Putative domains are

indicated. (B) Sequence

alignment of RNA binding

domains of A. thaliana GAR1-1

(At 3g03920, 201 aa), GAR1-2

(At5g18180, 189 aa) and NAF1

(At1g03530, 801 aa) proteins.

(C) Sequence alignment of

NAF1 protein of S. cerevisiae(492 aa), S. pombe (516 aa),

A. thaliana (801 aa), H. sapiens(494 aa) and D. melanogaster(564 aa) in the region of the

conserved NAF1 domain.

Residues that are identical in all

aligned sequences are shaded

Table 1 Coexpression of CBF5 and NAF1 genes with genes involved in RNA modification

Gene Gene description rs NAF1 rs CBF5

At2g37990 Ribosome biogenesis regulatory protein 0.6878 0.8661

At3g03920 GAR1 RNA binding protein 0.7161 0.8413

At5g08620 DEAD box RNA helicase 0.6937 0.8030

At3g11964 S1 RNA-binding domain- containing protein 0.6732 0.7977

At3g13570 SC35-like splicing factor 0.6516 0.7445

At5g08180 NHP2-like protein 0.6437 0.7269

At2g20490 Nucleolar RNA-binding NOP10 family protein 0.6030 0.6113

At4g30220 Small nuclear ribonucleoprotein F, putative 0.6052 0.6043

Data are extracted from the A. thaliana co-response database (http://csbdb.mpimp-golm.mpg.de). Values in columns 3 and 4 represent Sperman’s

rank order correlation coefficient rs in relation to NAF1 and CBF5. Putative components of the H/ACA box snoRNP complex are in bold

622 Plant Mol Biol (2007) 65:615–626

123

nucleolus (Fig. 5D). Due to the absence of unexpected

BiFC signal patterns, we assume that they are specific.

Moreover, BiFC experiments performed as a positive

control with two nuclear proteins resulted in BiFC signals

localizing exclusively in nucleoplasm (data not shown) and

never somewhere else in the cell. To find out whether the

nuclear bodies are identical with CBs, immunostaining

with anti-TMG and anti-GFP antibodies was performed on

nuclei of tobacco leaves transiently expressing CBF5 and

NAF1 BiFC constructs (Fig. 5E). Since anti-GFP anti-

bodies were raised against the full length recombinant

protein, it was expected that they can recognize restored

YFP as well as its parts in fusion with CBF5 and NAF1. In

most cases the GFP immunosignals were detected in

nucleoli, nucleoplasm and nuclear bodies (Fig. 5E).

Nuclear bodies stained with anti-GFP antibodies coincided

with anti-TMG stained snRNA. GFP immunosignals were

localized in the nucleoli of most nuclei analyzed, however,

in vivo BiFC fluorescence was rarely observed in nucleoli.

This indicates that CBF5 and NAF1 interact preferentially

within nucleoplasm and CBs rather than within the

nucleolus. To test separately the localization of CBF5 and

NAF1 proteins in N. benthamiana leaf nuclei, immuno-

staining with anti-c-Myc (labeling CBF5-NYFP) and anti-

HA (labeling NAF1-CYFP) antibodies was performed. In

most cases immunosignals detecting CBF5 were localized

within nucleoplasm, nuclear bodies and nucleoli (Fig. 5F)

like in A.thaliana, while three different localization pat-

terns were observed for NAF1 (Fig. 5G–I), similar to the

distribution of BiFC signals (Fig. 5B–D). Nuclear bodies

labeled with both, anti-c-Myc and anti-HA antibodies

coincided with strong TMG-snRNA signals (data not

shown). Control experiments in which AtCBF5-NYFP was

co-injected with unfused CYFP and AtNAF1-CYFP with

unfused NYFP did not show any fluorescence (data not

shown). Expression of the BiFC constructs in the negative

control experiments was also confirmed by immunostain-

ing (data not shown).

CBF5 is essential for viability of A. thaliana

To further examine the importance of AtCBF5, we iden-

tified and investigated two A. thaliana lines with disruption

of the CBF5 gene, atcbf5-1 and atcbf5-2, from the Syn-

genta collection of T-DNA insertion mutants. Both mutants

contain inverted T-DNA repeats with left border junctions

at their termini in 50non-coding region, 73 bp and 92 bp

upstream of the CBF5 coding region, respectively. The

T-DNA insertion generated deletion of two nucleotides in

Fig. 5 BiFC analysis showing interaction of AtCBF5-NYFP and

AtNAF1-CYFP within nuclei of transiently transformed N. benth-amiana leaves (the same results were obtained with AtNAF1-NYFP

and AtCBF5-CYFP constructs). (A) DIC image with BiFC signals,

(B–D) Three different patterns of BiFC signals; (B) signals dispersed

over nucleoplasm, (C) additionally clustered at a nuclear body (D)

additionally clustered at nuclear bodies and within the nucleolus (the

most rare case). (E) Colocalization of anti-GFP signals (red;

representing BiFC signals) and anti-TMG snRNA signals (green,

representing Cajal bodies). (F) Localization of CBF5 and (G–I) of

NAF1 in leaf nuclei after immunostaining with anti-Myc and anti-HA

antibodies, respectively. Arrows, nucleoli; arrow heads, Cajal bodies.

Bars = 20 lm for (A) and 2 lm for (B–I)

Plant Mol Biol (2007) 65:615–626 623

123

the atcbf5-2 allele. In both cases only hemizygous mutants

with normal vegetative development were obtained and

none of the plants was homozygous for the mutant allele.

We examined the segregation of the PPT resistance marker

of T-DNA tags of mutant alleles. All M2 lines showed a

segregation ratio close to 2:1 (resistant:sensitive) progeny.

PCR genotyping of 40 M3 resistant plants for each mutant

allele confirmed that they were heterozygous. Apparently,

a complete loss of AtCBF5 is lethal, indicating that CBF5

is essential for viability in A. thaliana.

Discussion

CBF5 is essential for yeast growth; spores with a disrupted

CBF5 gene divide only a few times, resulting in microcol-

onies of 4–20 cells (Jiang et al. 1993). Mutations in the gene

encoding the human homolog NAP57 are associated with

X-linked dyskeratosis congentia (Heiss et al. 1998). Obvi-

ously, also for A. thaliana CBF5 is essential for viability.

The high conservation of CBF5 implies an important

cellular role. The ability of Drosophila CBF5 to comple-

ment a CBF5 null mutation in yeast suggests that its

function is also conserved (Phillips et al. 1998). The

A. thaliana CBF5 protein was originally identified by

Maceluch et al. (2001) on the base of its high similarity to

the mammalian (67% identity and 81% similarity) and the

yeast (72% identity and 85% similarity, respectively)

proteins. However, up to now no functional characteriza-

tion of CBF5 was performed in Arabidopsis or other plants.

The localization of CBF5-EYFP within nucleoli and

nucleoplasm of A. thaliana and V. faba interphase nuclei

but not at mitotic chromosomes or their centromeres is

similar as described for mammals (Meier and Blobel 1994;

Heiss et al. 1999; Kukalev et al. 2005) and Drosophila

(Phillips et al. 1998). Nevertheless, antibodies directed

against a synthetic peptide derived from the putative barley

CBF5 recognized the centromeric regions on isolated

metaphase chromosomes of barley and V. faba (ten Hoopen

et al. 2000). That AtCBF5-EYFP does not recognize the

centromeres of V. faba might be due to differences between

both species regarding the region of CBF5 responsible for

the recognition of centromeres. Since the sequence of

V. faba CBF5 is currently not known, this hypothesis

cannot yet be tested. The absence of the CBF5-EYFP

signals on the centromeres of A. thaliana could be due to

the low amount of protein there which cannot be detected

by the applied techniques neither in vivo nor in situ.

The colocalization of CBF5 and TMG-capped snRNA in

CBs suggests that in A. thaliana, like in other organisms,

CBF5 is involved in modification of RNA.

We assume that the localization pattern of A. thaliana

CBF5-EYFP fusion protein is specific and not the result of

mislocalisation due to the overexpression using the CaMV

35S promoter, because (i) in most cases the CBF5-EYFP

fluorescence was observed in nucleolus and Cajal bodies

like in other organisms previously characterized (Meier

and Blobel 1994; Heiss et al. 1999), (ii) the localization of

CBF5 to the nucleoplasm, Cajal bodies and nucleoli are

consistent with CBF5 being one of the core proteins of

H/ACA box snoRNPs, (iii) dispersed nucleoplasmic

localization patterns were observed very rarely. Moreover,

nucleoplasmic localisation of CBF5 was also observed in

human cell culture using a CBF5/EGFP fusion construct

(Heiss et al. 1999) and anti-CBF5 antibodies (Kukalev

et al. 2005).

AtCBF5 showed interaction with the AtNAF1 protein in

the yeast two-hybrid system and in N. benthamiana. NAF1

of yeast interacts with CBF5 and NHP2, the core protein

components of mature H/ACA box snoRNPs (Ito et al.

2001) and human NAF1 binds HsCBF5 and shuttles

between nucleus and cytoplasm (Darzacq et al. 2006).

Depletion of NAF1 in yeast leads to a decrease in steady-

state accumulation of H/ACA box snoRNAs and of CBF5,

GAR1 and NOP10 (Dez et al. 2002).

Using antibodies against NAF1, Darzacq et al. (2006)

have shown that in cultured human cells NAF1 is localiz-

ing in nucleoplasm and is not enriched in CBs and nucleoli;

overexpressed NAF1-GFP fusion protein was found to be

accumulated in cytoplasm. In the same study, it was sug-

gested that NAF1 is interacting with CBF5 in cytoplasm

and nucleoplasm, where NAF1 is required for the H/ACA

box snoRNP maturation, while CBs and nucleoli contain

mature snoRNP complexes. Possibly, distribution of NAF1

and maturation of H/ACA box snoRNPs differ between

human and plants since AtNAF1 was preferentially local-

ized within nucleoplasm, very often in CBs and rarely in

nucleolus and in all these compartments it could interact

with CBF5 (Fig. 5C, D). In plants, assembly of H/ACA

box snoRNPs might take place also in CBs and in nucleoli

(Fig. 6). Alternatively, the intracellular NAF1 distribution

could be tissue and/or cell type-specific as shown for the

distribution of AtCBF5 in this paper or for HsCBF5 (Heiss

et al. 1999; Kukalev et al. 2005). The similarity of NAF1

and GAR1 domains in A. thaliana suggests that like in

human cells they might compete for the interaction with

CBF5 (Fig. 6). Moreover, according to the A. thaliana

nucleolar protein database (http://bioinf.scri.sari.ac.uk/

cgi-bin/atnopdb/home) a putative AtGAR1 protein

(At3g03920) fused to GFP was localized in the nucleolus,

nuclear bodies and the nucleoplasm. Also all other putative

components of H/ACA box snoRNP complex (GAR1,

NOP10 and NHP2) were found within nucleoli according

to proteomic analysis of A. thaliana nucleoli (Pendle et al.

2005). Additionally, coexpression of CBF5 and NAF1

genes with genes encoding GAR1, NOP10 and NHP2

624 Plant Mol Biol (2007) 65:615–626

123

suggests that the H/ACA box snoRNP complex of plants

might have the same protein components as that of yeast

and metazoa (Fig. 6). Interestingly, all protein components

of the H/ACA box snoRNP complex (GAR1, CBF5, NHP2

and NOP10) are very conserved among different organisms

over the entire length, while in the NAF1 protein, required

for the assembly of snoRNP complex, only the NAF1

domain is conserved. Comparison of domain structures of

A. thaliana NAF1 and of the previously characterised yeast

NAF1 protein (Fatica et al. 2002) revealed several differ-

ences (Fig. 4A). In A. thaliana NAF1 there is no RE/RS-

rich domain, which is present in yeast protein, and a

C-terminal Q-rich domain is present instead of the P + Q-

rich domain. Moreover, the Glu/Ser/Asp-rich domain

found in A. thaliana NAF1 was not found in any other

NAF1 proteins and NAF1 protein of A. thaliana is signif-

icantly larger than that of yeast and metazoa (see legend to

Fig. 4). Possibly NAF1 functions like an adaptor con-

necting the conserved H/ACA box snoRNP complex with

more variable species-specific components of the RNA

modification and RNA splicing machinery.

Although it is not clear how the nucleolar and CB

localization of CBF5 and its presumed function in RNA

modification could be related with role in centromere

function, we cannot exclude a centromere function of

CBF5 with certainty.

Acknowledgements We thank Andrea Kunze and Rita Schubert for

technical assistance, Bernhard Claus for help with confocal micros-

copy, Jorg Fuchs for flow-sorting of nuclei, Alice Navratilova for help

with examination of field bean hairy root cultures, Sabina Klatte and

Alexander Kukalev for helpful discussions, Karin Lipfert and Ursula

Tiemann for help with preparation of figures. This work was sup-

ported by a grant from the Deutsche Forschungsgemeinschaft to I.S.

(Schu 951/9-3).

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