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Research Article

Mapping a nucleolar targeting sequence of anRNA binding nucleolar protein, Nop25

Takashi Fujiwara, Shunji Suzuki⁎, Motoko Kanno, Hironobu Sugiyama, Hisaaki Takahashi,Junya TanakaDivision of Molecular and Cellular Physiology, Department of Molecular and Cellular Biology, School of Medicine,Ehime University, Toon City, Ehime 791-0295, Japan

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Present address: LabUniversity, Kofu, Yamanashi 400-0005, Japan

E-mail addre ss: suzukis@yama nashi.ac.jp

0014-4827/$ – see front matter © 2006 Elsevidoi:10.1016/ j.yexcr.2006.02.002

A B S T R A C T

Article Chronology:Received 8 November 2005Revised version received16 January 2006Accepted 2 February 2006Available online 3 March 2006

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription.The present study was undertaken to determine the mechanism of Nop25 localization inthe nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 tocontain a nuclear targeting sequence in the N-terminal and a nucleolar targetingsequence in the C-terminal. By expressing derivative peptides from the C-terminal asGFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide(KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transportedand fully retained in the nucleolus. When the peptide was fused with cMyc epitope andexpressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did notlocalize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion ofa subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine andarginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization.These results suggest that the lysine and arginine residue-enriched peptide is the mostprominent nucleolar targeting sequence of Nop25 and that the long stretch of basicresidues might play an important role in the nucleolar localization of Nop25. AlthoughNop25 contained putative SUMOylation, phosphorylation and glycosylation sites, theamino acid substitution in these sites had no effect on the nucleolar localization, thussuggesting that these post-translational modifications did not contribute to thelocalization of Nop25 in the nucleolus. The treatment of the cells, which expressed aGFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resultedin a complete dislocation of the protein from the nucleolus. These data suggested thatthe nucleolar targeting sequence might therefore play an important role in the binding ofNop25 to RNA molecules and that the RNA binding of Nop25 might be essential for thenucleolar localization of Nop25.

© 2006 Elsevier Inc. All rights reserved.

Keywords:Nop25Nucleolar proteinNucleolusNucleolar localizationRNA binding protein

oratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, Yamanashi. Fax: +81 55 220 8768. (S. Suzuki).

er Inc. All rights reserved.

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Introduction

The nucleolus is a plurifunctional “factory” in the cellnucleus which plays a role in ribosome biogenesis,including the transcription and processing of rRNA andthe assembly of ribosomal subunits [1]. Although thenucleolus is not enclosed by any membrane, it is structur-ally and functionally distinguished into three regions,namely fibrillar centers, dense fibrillar components andgranular components, thus suggesting that the nucleolus isa highly organized subcellular structure [2,3]. Recently, anexhaustive proteomic analysis of the human nucleolus hasbeen performed which thus provided us with a goodunderstanding of the nucleolar functions [4]. This impres-sive study demonstrated evidence that the nucleolus playsa role in more diverse biological phenomena, for example,in cell proliferation [5] and in the cell cycle [6], in additionto ribosome biogenesis. Owing to the complexity of thenucleolar functions, however, the complete picture offundamental mechanism of the nucleolus has yet to beelucidated.

We previously reported the molecular cloning andcharacterization of Nop25, which is a novel RNA bindingnucleolar protein and highly conserved in vertebratespecies [7]. Cytological studies using RNase A or actinomy-cin D and an immunoprecipitation experiment with anantibody against Nop25 demonstrated that Nop25 mightbind to rRNA molecules and that its nucleolar localizationmight also be associated with on-going rRNA transcription.Although further studies on the characterization of Nop25in the nucleolus have been carried out, so far we could notdemonstrate any crucial function that Nop25 plays in thenucleolus.

Several functional nucleolar targeting sequences ofwell-identified nucleolar proteins have been characterized[8–10]. The nucleolar localization of NF-κB inducingkinase is mediated by a stretch of basic amino acids,R–K–K–R–K–K–K [9]. In addition, an R/K–R/K–X–R/K motifhas also been found in various nucleolar proteins, suchas ribosomal protein S6, nucleophosmin and ARF tumorsuppressor protein [8]. Likewise, arginine-rich motifs forthe nucleolar localization of the viral proteins have beenidentified, for example, in Tat and Rev proteins ofhuman immunodeficiency virus 1 [11,12] and in Rexprotein of human T-cell leukemia virus [13]. Although abioinformatic study of human nucleolar proteins couldnot identify the consensus nucleolar targeting sequence,most nucleolar proteins seem to favor charged aminoacids, such as glutamic acid, aspartic acid, lysine andarginine [14].

In the present study, we report the mapping a nucleolartargeting sequence for the localization of Nop25 in thenucleolus. The nucleolar targeting sequence of Nop25mostly consists of lysine and arginine residues similar toother nucleolar targeting sequences. Furthermore, weherein demonstrate that the binding of Nop25 to RNAmolecules through a nucleolar targeting sequence mightplay an important role in the localization of Nop25 in thenucleolus.

Materials and methods

Chemicals

Hoechst 33258was obtained fromDojindo (Kumamoto, Japan).RNase A was purchased from Sigma (St. Louis, MO). Anantibody against the cMyc epitope tag and a Cy3-conjugatedanti-rabbit IgG were purchased from Affinity BioReagents(Golden, CO) and Chemicon (Temecula, CA), respectively. Arabbit polyclonal antibody against rat Nop25 was providedfrom Trans Genic Inc. (Kumamoto, Japan).

Construction of expression plasmids

A plasmid for expression of a GFP-fused Nop25 protein(designated as GFP-Nop25) was constructed according to aprevious report [7]. For the mapping of the nucleolar targetingsequence of Nop25, several deletion mutants were con-structed (Fig. 2A). To delete 70 amino acids from N-terminalof Nop25, PCR was performed with a GFP-Nop25 plasmid as atemplate. The nucleotide sequences of the primers were asfollows: 5′-GATCTCGAGCTATGCTGGCAGAAAGAGAGGA-3′containing an XhoI site (underlined) and 5′-GCAGAATTCT-CACTCCCCGTTGTGCCTGGCTT-3′ containing an EcoRI site(underlined). The amplified product was digested by XhoIand EcoRI followed by ligation into pEGFP-C1 (Clontech, PaloAlto, CA) which was digested by XhoI and EcoRI, thus resultingin GFP-Nop25ΔN70. The digestion of a GFP-Nop25 plasmidwith BamHI was performed to delete 60 amino acids from C-terminal of Nop25. After removing a BamHI-digested smallfragment, the digested plasmid was self-ligated again, thusresulting in GFP-Nop25ΔC60. To delete the amino acids fromboth the N- and C-terminals of Nop25, double deletion wasintroduced by deleting 60 amino acids of C-terminal from aGFP-Nop25ΔN70 plasmid with BamHI, thus resulting in GFP-Nop25ΔNC. The DNA fragments encoding 70 amino acids of N-terminal or 62 amino acids of C-terminal were amplified froma GFP-Nop25 plasmid by a PCR procedure with primer sets, 5′-GATCTCGAGCTATGGGCCGCAACAAGAAGAA-3′ containingan XhoI site (underlined) and 5′-GCAGAATTCCTTCAGGTA-CTCCTGGTGGC-3′ containing an EcoRI site (underlined) or 5′-GATCTCGAGCTCCCCTGCTCTCTCAGCGCATCT-3′ containingan XhoI site (underlined) and 5′-GCAGAATTCTCACTCCC-CGTTGTGCCTGGCTT -3′ containing an EcoRI site (underlined),respectively. The amplified products were digested by XhoIand EcoRI followed by ligation into pEGFP-C1 which wasdigested by XhoI and EcoRI, thus resulting in GFP-Nop25N70 orGFP-Nop25C62, respectively.

Construction of peptide-expression plasmids

To construct the expression plasmids for the expression ofNop25 peptides derived from 60 amino acids of C-terminal,sense and antisense oligonucleotides were annealed in 100mM NaCl under the following conditions: after incubation at99°C for 2 min, the reaction mixtures were gradually cooleddown from 72°C to 37°C for 2 h. The nucleotide sequences ofsense and antisense oligonucleotides used were as follows: 5′-TCGAGATCCCCTGCTCTCTCAGCGCATCTCCTCCCTCACAGCT-

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3′ and 5′-AATTAGCTGTGAGGGAGGAGATGCGCTGAGAGAG-CAGGGGATC-3′ for peptide1 (P1) encoding DPLLSQRISSLTA,5′-TCGAATCTCCTCCCTCACAGCTACACTGCATGCACA-CAGTCGG-3′ and 5′-AATTCCGACTGTGTGCATGCAGTG-TAGCTGTGAGGGAGGAGAT-3′ for peptide2 (P2) encodingISSLTATLHAHSR, 5′-TCGACGGAAGAAGGTCAAGAGGAAA-CACCCTCGGCGGGCGCAG-3′ and 5′-AATTCTGCGCCCGCC-GAGGGTGTTTCCTCTTGACCTTCTTCCG-3′ for peptide3 (P3)encoding RKKVKRKHPRRAQ, 5′-TCGAGCGCAGGACTCCACCAA-GAAACCTCCAAGTGCCACTCGT-3′ and 5′-AATTACGAGTGG-CACTTGGAGGTTTCTTGGTGGAGTCCTGCGC-3′ for peptide4(P4) encoding AQDSTKKPPSATR, 5′-TCGAACCAGCAAGACC-CAGCGCCGCCGTCGGATGACTGGAAAA3′ and 5′-AATTTTTTC-CAGTCATCCGACGGCGGCGCTGGGTC2TTGCTGGT-3′ forpeptide5 (P5) encoding TSKTQRRRRMTGK, 5′-TCGAAAGAG-GAAACACCCTCGGCGGGCGCAGGACTCCACCAAGAAACCTCCA-3′ and 5′-AATTTGGAGGTTTCTTGGTGGAGTCCTGCGCCCGCC-GAGGGTGTTTCCTCTT-3′ for peptide6 (P6) encodingKRKHPRRAQDSTKKPP, 5′-TCGAAAGAAACCTCCAAGTGC-CACTCGTACCAGCAAGACCCAGCGCCGCCGTCGG-3′ and 5′-AATTCCGACGGCGGCGCTGGGTCTTGCTGGTACGAG-TGGCACTTGGAGGTTTCTT-3′ for peptide7 (P7) encodingKKPPSATRTSKTQRRRR, 5 ′-TCGAAAGAGGAAACACC-CTCGGCGGGCGCAGGACTCCACCAAGAAACCTCCAA-GTGCCACTCGTACCAGCAAGACCCAGCGCCGCCGTCGG-3′ and5′-AATTCCGACGGCGGCGCTGGGTCTTGCTGGTACGAGT-GGCACTTGGAGGTTTCTTGGTGGAGTCCTGCGCCCGCC-GAGGGTGTTTCCTCTT-3′ for peptide8 (P8) encodingKRKHPRRAQDSTKKPPSATRTSKTQRRRR. The annealing pro-ducts were ligated into the XhoI–EcoRI site in pEGFP-C1, thusresulting in expression plasmids, GFP-P1, GFP-P2, GFP-P3, GFP-P4, GFP-P5, GFP-P6, GFP-P7 and GFP-P8, respectively (Fig. 3A).

To express a cMyc epitope (EQKLISEEDL)-tagged P8 peptidein the cells, PCRwas performedwith a GFP-Nop25 plasmid as atemplate. The nucleotide sequences of the primers used were asfollows: 5′-CTGGCTAGCAAGAGGAAACACCCTCGGCG-3′ containingan NheI site (underlined) and 5′-CGCCTCGAGTCACAGATCCTCTTCT-GAGATGAGTTTTTGTTCCCGACGGCGGCGCTGGGTCT-3′ containingan XhoI site (underlined) and encoding the cMyc epitope(italic). The amplified product was digested withNheI and XhoIand ligated into the NheI–XhoI site in pcDNA3.1 (Invitrogen,Groningen, Netherlands), thus resulting in P8-cMyc.

Construction of deletion plasmids

Deletion of P8 or a subdomain (KRKHPRRAQ) in P8 from a GFP-Nop25 plasmid was performed using a PCR procedure withprimer sets, 5′-GACCTTCTTCCGACTGTGT-3′ and 5′-ATGACTG-GAAAAGCCAGGCA -3′ for deletion of P8 or 5′-GACCTTCTTCC-GACTGTGT-3′ and 5′-GGACTCCACCAAGAAACCT-3′ fordeletion of the subdomain, respectively. The amplifiedproducts were self-ligated, thus resulting in GFP-Nop25ΔP8or GFP-Nop25ΔKRKHPRRAQ, respectively (Fig. 4A).

Amino acid substitution

Amino acid substitution, from lysine and arginine residues toalanine residues in the subdomain (KRKHPRRAQ), was per-formed using a PCR procedure. Primer sequences usedwere asfo l lows : 5 ′ -AGCTGCACACCCTGCAGCA-3 ′ and 5 ′ -

GCGACCTTCTTCCGACTGTGT-3′. The amplified product wasself-ligated, thus GFP-Nop25AAAHPAAAQ (Fig. 4A). Aminoacid substitution was also introduced in putative SUMO-targeting motifs and phosphorylation sites of a P8 peptide(Fig. 5). Sense and antisense oligonucleotides were annealedas described above and the annealing product was ligated intothe XhoI–EcoRI site in pEGFP-C1, thus resulting in GFP-mutatedP8. The nucleotide sequences of oligonucleotides usedwere asfollows: a sense oligonucleotide, 5′-TCGAAAGAGGGCA-CACCCTCGGCGGGCGCAGGACGCCGCCAAGGCACCTC-CAAGTGCCACTCGTGCCAGCAAGGCCCAGCGCCGCCGTCGG-3′and an antisense oligonucleotide, 5′-AATTCCGACGGCGGC-GCTGGGCCTTGCTGGCACGAGTGGCACTTGGAGGTG-CCTTGGCGGCGTCCTGCGCCCGCCGAGGGTGTGCCCTCTT-3′.

Transfection

COS7 cells were maintained in Dulbecco's modified Eagle'smedium supplemented with 10% (v/v) heat-inactivated fetalbovine serum at 37°C in a CO2 incubator and split in freshmedia every 3–4 days. The transfection of expression plasmidsin COS7 cells was performed using a PerFectin transfectionregent (Gene Therapy Systems, San Diego, CA). Observationswere then carried out 24 h after transfection.

RNase A treatment

RNase A treatment was done according to the method ofSuzuki et al. [7]. Briefly, the cells were fixed in methanol for 2min at −20°C and then incubated in 1 ng/ml RNase A for 2 h atroom temperature.

Indirect immunofluorescence analysis

An indirect immunofluorescence analysis was performedaccording to the method of Kanno et al. [15]. Briefly, thecells grown on coverslips were fixed with paraformaldehydefollowed by treatment with Triton X-100 and NP-40. Thecells were incubated with antibodies against Nop25 or cMycepitope and then with a Cy3-conjugated secondary anti-body. After washing the cells, the cells were stained withHoechst 33258.

Observations

Observations of the cells tested were carried out under oilimmersion (100× objective magnification) using a differential-interference-contrast (DIC) and fluorescencemicroscope, BX61(Olympus, Melville, NY). Photoshop 5.5 (Adobe Systems Inc.,San Jose, CA)wasused for the correction and adjustment of thebrightness and contrast of figures.

Results

Nucleolar localization of Nop25

Rat Nop25 encodes the 216 aa protein, with a calculatedmolecular weight of 25.2 kDa and an theoretical pI of 10.07(accession no. AY917132). Both the N- and C-terminals ofNop25 contain many basic residues such as lysine and

Table 1 – Prediction of nuclear targeting sequences inNop25 by PSORT II

Four residue pattern fornuclear targeting

Seven residue pattern fornuclear targeting (Robbins and

Dingwall consensus)

Position: 5 KKKK Position: 14 RRPRLILNFDEEKRREYPosition: 6 KKKR Position: 26 KRREYLTGFHKRKVERKPosition: 14 RRPR Position: 27 RREYLTGFHKRKVERKKPosition: 35 HKRK Position: 190 KKPPSATRTSKTQRRRRPosition: 178 KRKHPosition: 203 RRRR

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arginine, which are favored within the nuclear proteins (Fig.1A). When Nop25 was expressed as a GFP-fusion protein inCOS7 cells, Nop25 was localized in the nucleolus (Fig. 1B). Thenucleolar localization of endogenous Nop25 in COS7 cells wasalso proven by an indirect immunofluorescence analysis usingan antibody against Nop25 (Fig. 1C). As a result, Nop25 is thusconsidered to belong to a nucleolar protein family as describedpreviously [7].

Mapping the nucleolar targeting sequence of Nop25

The scanning of Nop25 amino acid sequence using PROSITE, adatabase of the protein families and domains available at http://kr.expasy.org/prosite/ [16], demonstrated that Nop25 containsthree bipartite nuclear targeting sequences (aa 14–30;RRPRLILNFDEEKRREY, aa 26–42; KRREYLTGFHKRKVERKK, andaa 190–206; KKPPSATRTSKTQRRRR). Furthermore, based on asearch of the Nop25 amino acid sequence using programsdesigned to predict the protein sorting signals and the localiza-tion sites (PSORT II, available at http://psort.ims.u-tokyo.as.jp/),Nop25 was thus found to have several predicted nucleartargeting sequences in both the N- and C-terminals (Table 1).

Nop25 proteins truncated at N- and/or C-terminal wereexpressed as GFP-fusion proteins in COS7 cells (Fig. 2). Thedeletion of 70 amino acids from N-terminal had no effect onnucleolar localization (Fig. 2B, GFP-Nop25ΔN70). In contrast toN-terminal deletion, a Nop25 protein deleted 60 amino acidsfrom C-terminal failed to localize in the nucleolus, although itwas still localized in the nucleus (Fig. 2B, GFP-Nop25ΔC60). Bydouble deletion at both the N- and C-terminals, the deletedNop25 proteinwas neither localized in the nucleolus nor in thenucleus (Fig. 2B, GFP-Nop25ΔNC). GFP-Nop25ΔC60 and GFP-Nop25ΔNC did not lead the complete exclusion of the GFP

Fig. 1 – Nucleolar localization of Nop25. (A) The deduced aminoand arginine residues are shown in red. (B) A GFP-fusion proteinCOS7 cells (GFP-Nop25). The green color showed the fluorescencNop25 was also proven, when endogenous Nop25 in COS7 cellsCy3-conjugated secondary antibody (anti-Nop25). The red color sstain the nucleoplasm of the cells (Hoechst). The arrows indicate

signals from the nucleolus (Fig. 2B). Similar localization of aGFP protein in the nucleolus was observed in the GFP-expressed control cells (Fig. 2B, GFP), suggesting that thenucleolar localization of the deletedmutants might be artifactdue to the overexpression of a GFP protein. As expected, a GFP-fusion protein with 62 amino acids of the C-terminal waslocalized in the nucleolus (Fig. 2B, GFP-Nop25C62). Thesubcellular location of a GFP-fusion protein tagged with 70amino acids of the N-terminal was predominantly in thenucleus, although the nucleolar localization was partiallyobserved (Fig. 2B, GFP-Nop25N70).

Taken together, these results suggested the nucleolartargeting sequence(s) of Nop25 to be in the C-terminal whilethe N-terminal of Nop25 might function as another nucleartargeting sequence.

Nucleolar targeting sequence in C-terminal of Nop25

To determine the crucial nucleolar targeting sequence(s) inthe C-terminal of Nop25, small peptides derived from 62amino acids of C-terminal were fused with a GFP protein and

acid sequence of rat Nop25 (accession no. AY917132). Lysinewith Nop25 was localized in the nucleolus of the transfectede of the GFP-fusion protein. (C) The nucleolar localization ofwas immunostained with an antibody against Nop25 and ahowed the fluorescence of Cy3. Hoechst 33258 was used tonucleoli in the nucleus. Scale bar, 10 μm.

Fig. 2 – Mapping a nucleolar targeting sequence of Nop25. (A) A schematic representation of the deletion mutants of the GFP-fusion proteins with Nop25. (B) The deletion mutants were transfected and expressed in the COS7 cells. g, GFP fluorescence. h,Hoechst staining. d, an image observed using a DIC microscope. Scale bar, 10 μm.

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expressed in the COS7 cells (Fig. 3A). P1, P2, P4, P5 or P7-fusedGFP proteins were dispersed into the cytoplasmic space (Fig.3B, GFP-P1, -P2, -P4, -P5 and -P7). P3 or P6-fused GFP proteinswere predominantly localized in the nucleus. Although thenucleolar localization of the GFP fluorescent signal was found

in the cells transfectedwith GFP-P3 or -P6, the signalsmight benot firmly retained in the nucleolus and thus diffused into thenucleus or, occasionally, into cytosolic space (Fig. 3B, GFP-P3and -P6). P8-fused GFP protein thus showed the nucleolarlocalization (Fig. 3B, GFP-P8). When the cMyc epitope was

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expressed as a fusion protein with P8 in COS7 cells, the cMycepitopewas predominantly localized in the nucleolus (Fig. 3C),suggesting that P8 might be a nucleolar targeting sequence ofNop25.

Fig. 3 – Nucleolar targeting sequence in C-terminal of Nop25. (A) TNop25. A schematic representation of small peptides (P1–P8) wasto P8 peptides were tagged to a GFP protein and expressed in COclassified into three groups such as nucleolar, nuclear and cytosolGFP-fusion proteins with P1–P8 peptides. (C) The nucleolar localiexpressed as a fusion proteinwith a P8 peptide in COS7 cells. Theepitope and a Cy3-conjugated secondary antibody (anti-cMyc). H(Hoechst). The red color showed the fluorescence of Cy3. Merge,

Furthermore, mutation experiments were performed (Fig.4). When P8, a putative nucleolar targeting sequence, wascompletely deleted from Nop25, the mutated Nop25 proteinwas not localized in the nucleolus (Fig. 4B, GFP-Nop25ΔP8).

hemapping of a nucleolar targeting sequence in C-terminal ofshown. Lysine and arginine residues are shown in red. (B) P1S7 cells (GFP-P1 to -P8). The subcellular localization could beic localization. The green color showed the fluorescence of thezation of a P8 peptide-tagged cMyc. The cMyc epitope wascells were immunostainedwith an antibody against the cMycoechst 33258 was used to stain the nucleoplasm of the cellsa merged image. Scale bar, 10 μm.

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Interestingly, Nop25 lost the nucleolar localization by deletionof a subdomain (KRKHPRRAQ), which was overlapped amongP3, P6 and P8 (Fig. 4B, GFP-Nop25ΔKRKHPRRAQ). When basicresidues, lysine and arginine, in the subdomain were substi-tuted to alanine residues, the mutated Nop25 protein waslocalized in the nucleus, but not particularly in the nucleolus(Fig. 4B, GFP-Nop25AAAHPAAAQ).

Taken together, these results suggest that a P8 peptide,which has a long stretch of basic residues, is the mostprominent nucleolar targeting sequence and that the longstretch of basic residues might play an important role in thenucleolar localization of Nop25.

Post-translational modification of Nop25 might not contributeto the nucleolar localization

Based on the amino acid sequence analysis of a P8 peptide, anucleolar targeting sequence of Nop25, using the PROSITE andSUMOplot Prediction Program (available at http://www.abgent.com/sumoplot.html), P8 was found to have fourphosphorylation sites for protein kinase C (PKC) and twoSUMO-targeting motifs (Fig. 5). The SUMOylation of a certainprotein by SUMOs is one type of post-translational modifica-tion which affects the subcellular localization of the protein[17–19]. The SUMOylation targets lysine in the consensussequence ØKXE (Ø is a hydrophobic residue) of substrateproteins [18,20]. To determine whether these post-transla-

Fig. 4 – Requirement of the basic residues in a nucleolar targeschematic representation of the mutants of the GFP-fusion proteexpressed in COS7 cells. The green color showed the fluorescenc

tional modifications are associated with the nucleolar local-ization of Nop25, serine and threonine residues targeted byprotein kinase C and lysine residues targeted by SUMO weresubstituted with alanine residues, thus resulting in mutatedP8 (Fig. 5). When a GFP-fusion protein with mutated P8 wasexpressed in COS7 cells, the GFP fluorescent signals were stilllocalized in the nucleolus (Fig. 5). Furthermore, the pointmutation experiment of the putative post-translational mod-ification was performed using full-length Nop25. Using thePROSITE and SUMOplot Prediction Program, Nop25 was foundto have eight PKC phosphorylation sites (90TAK92, 159SQR161,173SRK175, 188STK190, 189TKK191, 198TSK200, 201TQR203and 208TGK210), one N-glycosylation site (102NHTV105), fourcasein kinase II phosphorylation sites (109TVSD112,111SDID114, 134SQEE137 and 140SSME143), one cAMP- andcGMP-dependent protein kinase phosphorylation site(205RRMT208) and six SUMO-targeting motifs (7KKRD10,37RKVE40, 53LKQE57, 91AKTE94, 179RKHP182 and190KKPP193). When amino acid substitution of all residuestargeted by enzymes to alanine residues was introducedin full-length Nop25 and expressed as a GFP-fusion proteinin COS7 cells, the GFP fluorescent signals of all mutantswere still localized in the nucleolus (data not shown). Thesefindings suggested that the putative SUMO-targetingmotifs, phosphorylation sites and a glycosylation site foundin Nop25 might not contribute to the nucleolar localization ofNop25.

ting sequence for the nucleolar localization of Nop25. (A) Ains with Nop25. (B) The mutants were transfected ande of the GFP-fusion protein. Scale bar, 10 μm.

Fig. 6 – The subcellular localization of a P8 peptide in the cellstreated with RNase A. (A) The subcellular localization ofendogenous Nop25. COS7 cells were treated with RNase A

Fig. 5 – The SUMO-targeting motifs and phosphorylationsites do not contribute to the nucleolar localization of Nop25.Position of the putative SUMO-targeting motifs andphosphorylation sites in a P8 peptide was shown in the leftpanel. The upper sequence indicated amino acid residuescomprising a wild-type P8 peptide. Putative SUMO-targetingmotifs were underlined. The lysine residues targeted bySUMOs and serine and threonine residues targeted byprotein kinase C were shown in bold. The lower sequenceindicated amino acid residues comprising a mutated P8peptide. The substituted alanine residues were shown in redand underlined. Wild-type P8 or mutated P8 was expressedas GFP-fusion proteins in COS7 cells (GFP-P8 or GFP-mutatedP8, respectively). The green color showed the fluorescenceof the GFP-fusion proteins. Scale bar, 10 μm. (Forinterpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

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A nucleolar targeting sequence may bind to RNA molecules inthe nucleolus

Nop25 binds to RNA molecules, probably to the 28S rRNA, inthe nucleolus [7]. To evaluate whether a nucleolar targetingsequence binds to RNA molecules, we investigated thelocalization of a GFP-fusion protein with a P8 peptide inRNase A-treated COS7 cells. After treating non-transfectedCOS7 cells with RNase A, endogenous Nop25 was found tobe completely dislocated from the nucleolus, reformingsmall speckles of endogenous Nop25 in the nucleoplasm(Fig. 6A, [7]). In the transfected cells with a GFP-P8 plasmid,the treatment of the cells with RNase A induced a similardislocation of the GFP-fusion protein with P8 from thenucleolus (Fig. 6B), thus suggesting that a nucleolar target-ing sequence may bind to RNA molecules in the nucleolus.However, the reformation of the dislocated protein wasdifferent from that of endogenous Nop25 (Fig. 6). Based onthis finding, the binding of Nop25 to RNA moleculesthrough a nucleolar targeting sequence might thus playan important role in the localization of Nop25 in thenucleolus.

and immunostained with an antibody against Nop25 and aCy3-conjugated secondary antibody (Nop25). The red colorshowed the fluorescence of Cy3. (B) The subcellularlocalization of a P8 peptide. COS7 cells were transfected witha GFP-P8 expression plasmid and then were treated withRNaseA (GFP-P8). The green color showed the fluorescence ofthe GFP-fusion protein. Hoechst 33258 was used to stain thenucleoplasm of the cells (Hoechst). Scale bar, 10 μm.

Discussion

The above study demonstrated the mapping of a nucleolartargeting sequence of Nop25. Lysine and arginine residue-enriched sequence (termed as P8 in the present study) in C-terminal functioned as a nucleolar targeting sequence.

Furthermore, this sequence might also play a role in thebinding of Nop25 to RNA molecules.

The nucleolar targeting (localization) sequences wereinvestigated in well-known nucleolar proteins. For example,an R/K–R/K–X–R/K motif for ribosomal protein S6, nucleo-phosmin, and ARF tumor suppressor protein [8], a R–K–K–R–K–K–K sequence for NF-κB inducing kinase [9] and arginine-richmotifs for viral proteins [11–13] were identified as nucleolartargeting sequences. Likewise, the sequence to localize a novelnucleolar protein Nop25 in the nucleolus contained a lot oflysine and arginine residues (KRKHPRRAQDSTKKPP-SATRTSKTQRRRR). In line with the finding of a bioinformaticstudy of nucleolar proteins [14], small cluster comprised bycharged amino acids, such as lysine and arginine, seem to

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clearly play a central role in the nucleolar targeting ofnucleolar proteins.

Although the nucleolus is a stable compartment in thenucleus, most of its components including nucleolarproteins rapidly move from the nucleolus to the nucleo-plasm [21]. For example, the fluorescence recovery afterphotobleaching (FRAP) and the fluorescence loss in photo-bleaching (FLIP) analyses demonstrated that the retentiontimes of the tested nucleolar proteins in the nucleolus to beon the order of tens of seconds [22,23]. Furthermore, it hasbeen estimated that the retention of nucleolar proteins inthe nucleolus depends on their affinity against nucleolarcomponents, for example interactions with other nucleolarproteins or RNA molecules [24]. A previous report demon-strated that Nop25 could bind to RNA molecules, especially28S rRNA, and that treatment of the cells with RNase Aor actinomycin D resulted in the dispersion of Nop25 outof the nucleolus [7]. In addition, we herein showed thatthe fluorescence of GFP was dispersed from the nucleolus tothe nucleoplasm in the RNase A-treated cells expressing aGFP-fusion protein with a nucleolar targeting sequence ofNop25 (Fig. 6). When lysine and arginine residues of thesubdomain (KRKHPRRAQ) in the nucleolar targeting se-quence were substituted to alanine residues, the mutantprotein was not particularly localized in the nucleolus(Fig. 4B, GFP-Nop25AAAHPAAAQ). Taken together, thesefindings suggest that the longest peptide, which has along stretch of basic residues, is the most prominentnucleolar targeting sequence of Nop25 and seem to indicatethat the retention of Nop25 in the nucleolus might dependon the binding of Nop25 with the RNA molecules throughthe long stretch of basic residues in the nucleolar targetingsequence. Further studies are necessary to prove thebinding of the nucleolar targeting sequence with RNAmolecules in the nucleolus.

The phosphorylation of nucleolar proteins is one mech-anism to transport the proteins into the nucleolus [25,26]. Forexample, the nucleolar localization of promyelocytic leuke-mia tumor-suppressor protein (PML) after DNA damageoccurred was dependent on the phosphorylation of PML byATR kinase [26]. In contrast, the phosphorylation of anucleolar protein through a nucleolar retention motif hasbeen reported to stimulate the export of the nucleolarprotein to the nucleus [27]. Regarding the nucleolar targetingof Nop25, the amino acid substitution of putative phosphor-ylation sites of the sequence had no effect on the nucleolarlocalization (Fig. 5). In addition, SUMOylation, which trans-ports the nuclear proteins to the nucleus [17–19], did notcontribute to the nucleolar localization of Nop25, since theamino acid substitution in the putative SUMO-targetingmotifs had no effect on the subcellular localization (Fig. 5).Similar results were provided with full length Nop25 (datanot shown). Although the positions of the phosphorylationor SUMOylation on Nop25 remain to be defined, we foundNop25 to be phosphorylated while a SUMO-1 protein was co-precipitated with a Nop25 protein based on the findings of animmunoprecipitation experiment (Suzuki et al., in prepara-tion). Further investigation on the phosphorylation andSUMOylation of Nop25 may thus provide new informationon the function of Nop25 in the nucleolus.

In conclusion, we identified a nucleolar targeting se-quence of Nop25. This sequence has a long stretch of basicamino acids, lysine and arginine, and might play animportant role in the binding of Nop25 to RNA molecules.Moreover, the RNA binding of Nop25 through the sequencemay also be essential for the localization of Nop25 in thenucleolus. One important question that remains to beclarified is what does Nop25 do in the nucleolus? To answerthis question, experiments using Nop25-knockout mice bythe conditional gene disruption or Nop25-knockdown celllines by an RNA interference technique are required. Furtherstudies are now under way to determine the precisebiological function of Nop25 in the nucleolus.

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