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Original Paper	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 893-900
doi:10.1111/j.1745-7270.2008.00472.x

Expression and characterization of rice putative PAUSED gene

Chengguo Yao^1,3#, Liangfa Ge^2,3#, Wei Li^1,3, Botao Zhao^1,3, Chaoqun Li⁴, Kangcheng Ruan^1,3*, Hongxuan Lin^2,3, and Youxin Jin^1,3*

¹ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai 200031, China

² State Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai 200032, China

³ Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

⁴ Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China

Received: May 08, 2008�������

Accepted: August 31, 2008

This work was supported by grants from the National Natural Sciences Foundation of China (No. 30430210), the National Key Basic Research� and Development Program (No. 2005CB724602) and the Chinese Academy of Sciences (KSCX1-YW-R-64, KSCX2-YW-R-096)#These authors contributed equally to this work

*Corresponding author:

Youxin Jin: Tel, 86-21-54921222; Fax, 86-21-54921011; Email, [email protected]

Kangcheng Ruan: Fax, 86-21-54921011; Email, [email protected]

In Arabidopsis, PAUSED (PSD) encodes the ortholog of los1p/exportin-t, which mediates the nuclear export of transfer RNA (tRNA) in yeast and mammals. However, in monocot plants such as rice, knowledge of the corresponding ortholog is limited, and its effects on growth development and productivity� remain unknown. In this study, we verified a rice transfer-DNA insertional mutant psd line and analyzed its phenotypes; the mutant displayed severe morphological defects including retarded development and low fertility compared with wild-type rice. Examining intronless tRNA-Tyr and intron-containing� pre-tRNA-Ala expression levels in cytoplasmic and nuclear fraction with Northern blot analysis between wild-type and mutant leaf tissue suggested that rice PSD might be involved in tRNA export from the nucleus to the cytoplasm. Additionally, reverse transcription-polymerase chain reaction� analysis revealed that PSD transcript was expressed throughout normal rice plant development, and subcellular localization assays showed that rice PSD protein was present in both the nucleus and cytoplasm. In summary, our data implied� that the putative PSD gene might be indispensable for normal rice development and its function might be the same as that of Arabidopsis PSD.

Keywords������� PAUSED; tRNA export; development defects; rice

The transport of different RNA species from the nucleus to the cytoplasm is fundamental to gene expression. A general� paradigm has been established in which RNA are exported through the nuclear pore complexes via mobile export receptors after being produced in the nucleus. Small RNA, such as transfer RNA (tRNA) and microRNA, follow� a relatively simple pathway by directly binding to export receptors, while large RNA, such as rRNA and mRNA, are assembled into complicated ribonucleoprotein with exporters or other specific adaptor proteins [1,2].

As a member of the karyopherin superfamily, exportin-t is the principal tRNA exporter in vertebrate cells that binds to tRNA in a typical Ran-GTPase-dependent manner. Properly� processed 5� and 3� termini, secondary and tertiary� structural elements of tRNA, are decoded by exportin-t, meaning that tRNA quality control is performed before export [3-5]. In plants, Arabidopsis PSD is the ortholog of exportin-t and may function as tRNA exporter [6]; the psd mutant displays a series of growth and development defects, including delayed leaf production and abnormal inflorescence morphology. Rice is a model monocot plant that differs from Arabidopsis, which is representative of dicot plants. The expression pattern of PSD homolog and its role in normal rice plant development remain unknown. In this study, we employed a rice transfer-DNA (T-DNA) insertional mutant to explore rice PSD to gain a more comprehensive� understanding of plant PSD gene annotation� and to verify the diversity and complexity of pathways for plant tRNA export [7].

Materials and Methods

Plant culture

Wild-type rice (Oryza sativa L. subsp. Japonica cv. Dongjin) and T2 generation T-DNA insertional line (No. 1B-01410) seeds were obtained from Plant Functional Genomics Laboratory (Kyoungbuk, Korea) [8-11]. The seeds were germinated first and then transferred to farmland� belonging to the Shanghai Academy of Agricultural� Sciences (Shanghai, China) in order to adapt to natural growth conditions.

Sequence annotation and phylogenetic analysis

PSD gene sequences were downloaded from the NCBI gene database. Exons were indicated with Vector NTI (Invitrogen, Carlsbad, USA). Protein sequences showing obvious identity were downloaded from the rice PSD BLAST result in the NCBI protein database (http://www.ncbi.nlm.nih.gov/blast). Sequences were aligned using Clustalx1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) and further adjusted manually in BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html) [12,13]. Phylogenetic analysis� was done using the maximum likelihood method by tree-puzzle-5.2 (http://www.tree-puzzle.de/) [14].

Polymerase chain reaction (PCR) verification of the T-DNA insertional mutant

Leaf genomic DNA was purified using traditional CTAB method [15]. Two common PCR were performed for the differentiation of wild-type, heterozygote and homozygote plants. The primers were designed using the NCBI database� and web service (http://signal.salk.edu/cgi-bin/RiceGE). Primers for rice genome (PCR product: 409 bp) are 5�-TGCAGCTTATTTTCATTCA-3� (forward) and 5�-GGCATTTGCTGAAGATTTA-3� (reverse); primers for the T-DNA genome (PCR product: 543 bp) are 5�-TCA�GCC�A�T�TGGATCATAGT-3� (forward) and 5�-CCTG�TA�A�G�A�T�TTAGCACCC-3� (reverse). In brief, the PCR was conducted� for 30 cycles in a thermal controller (PTC-100; Bio-Rad, Hercules, USA). Each amplification cycle consisted of 0.5 min at 94 �C for denaturizing, 0.5 min at 55 �C for primer annealing and 1 min at 72 �C for extension.

Reverse transcription (RT)-PCR assay

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, USA). Initially, 2 mg total RNA was reverse transcribed� with gene-specific downstream primer by Moloney murine leukemia virus (M-MLV) reverse transcriptase� (Promega, Madison, USA) in a total volume of 25 ml, and cDNA was generated at 37 �C for 30 min. Expression level was determined using Taq PCR MasterMix system (Tiangen, Beijing, China) in a total volume of 25 ml reaction mixture containing 12.5 ml 2�master mix, 0.5 ml (20 mM) sense primer, 0.5 ml (20 mM) antisense primer, 2 ml cDNA template and 9.5 ml distilled water. The PCR was then performed for 28 amplification cycles in the thermal controller (PTC-100; Bio-Rad). Each cycle consisted of 0.5 min at 94 �C for denaturizing, 0.5 min at 56 �C for primer annealing and 1 min at 72 �C for extension. Primers� were used as follows: Actin (PCR product: 336 bp): 5�-TCCATCTTGGCATCTCTCAG-3� (forward) and 5�-G�T�A�CCCTCATCAGGCATCTG-3� (reverse); PSD (PCR product: 126 bp): 5�-cgacagttgctcgttgat-3� (forward) and 5�-TGCGATGGCAGGAAACAC-3� (reverse).

Subcellular localization assay

The full coding sequence used for transient expression of PSD in onion epidermal cells was amplified from full length cDNA clone AK099895 using the forward primer 5�-GGATCCGACGACCTCGAGCAGGCCAT-3� and reverse primer 5�-GAGCTCCTATCTGAAGACAAGACTCC-3�. The PCR product was subcloned into the BamHI/SacI site of pA7-green fluorescent protein (GFP) expression vector� under control of the enhanced cauliflower mosaic virus 35S promoter. The recombinant pA7-GFP-PSD plasmid and pA7-GFP plasmid (used as control) were separately transferred by bombardment into onion epidermal cells using a gene gun (PDS-1000/He; Bio-Rad) according to the instruction manual. Transformed cells were examined using a confocal microscope (Olympus FV500, Tokyo, Japan) after incubation at 25 �C for 24 h on Murashige and Skoog medium.

Cellular fractionation

Cellular fractionation was performed as described [6]. Briefly, leaf tissue from 4-week-old seedings were frozen in liquid nitrogen and ground to a powder with a mortar and pestle. Cell wall-disrupting buffer [10 mM potassium phosphate (pH 7.0), 100 mM NaCl, 10 mM 2-mercaptoethanol, 1 M hexylene glycol] was then added to make a thick slurry. This mixture was filtered through Miracloth (Calbiochem, San Diego, USA) to remove large chunks of tissue and centrifuged at 1500 g for 10 min at 4 �C to pellet nuclei and cell debris. After centrifugation, the supernatant was collected and recentrifuged at 13,000 g for 15 min at 4 �C. The supernatant of this second centrifugation� was saved for the cytoplasmic fraction. The first pellet was washed with nuclei preparation buffer [10 mM potassium phosphate (pH 7.0), 100 mM NaCl, 10 mM 2-mercaptoethanol, 1 M hexylene glycol, 10 mM MgCl2, 0.5% Triton X-100] and centrifuged at 1500 g for 10 min at 4 �C. After centrifugation, the supernatant was discarded and the pellet was washed with nuclei preparation buffer. Washing and centrifugation were repeated four to five times, and the final pellet was saved for the nuclear fraction. RNA was extracted from the cytoplasmic and nuclear fractions with TRIzol reagent [16].

Northern blot analysis for tRNA

A total of 10 mg cytoplasmic and 2 mg nuclear RNA were subjected to electrophoresis on an 8 M urea/12% denaturing� polyacrylamide gel, transferred to Hybond N+ membranes (Amersham Pharmacia, Uppsala, Sweden) and then hybridized� with [g-32P]-labeled probes. Oligonucleotide probes were labeled with T4 polynucleotide kinase (MBI Fermentas, St Leon-Rot, Germany). Hybridization and washing were carried out at 40 �C in the ExpressHyb Hybridization� Solution (Toyobo, Tokyo, Japan) according to the manufacturer�s instructions. The membrane was air-dried and then measured by phosphorimager (Amersham Pharmacia). 5S rRNA served as the loading control for the quantitative calculation, and U6 small nuclear RNA was used as marker of nuclear RNA. Probe sequences were as follows: 5S rRNA: 5�-AGGACTTCCCAGGAGGTCACCC-3�; U6 RNA: 5�-TCGATTTGTGGGTGTCAT-3�; tRNA-tyr: 5�-ACCTGCCGGATTCGAACCAGCG-3�; pre-tRNA-Ala: 5�-CAAGAATGGGATTCGAACCCA-3�.

Results

Genomic organization of rice putative PSD

Los1p/exportin-t proteins form a large family among many species, such as yeast, animals and plants, containing xpo1 domain in the primary structure [17,18]. By searching the rice genome database, we found that only one homolog shares 51% sequence identity with Arabidopsis PSD according� to sequence alignment in NCBI. The homolog�s existence was previously validated by rice full-length complementary DNA project (GenBank accession No. AK099895; http://cdna01.dna.affrc.go.jp/cDNA/). Rice psd is located in chr07 (25238070-25246227). The AUG translation start site and UAG stop codon are respectively located at 151 and 3091 bp of the AK099895 clone. As shown in Fig. 1(A), the rice PSD protein-encoding region is comprised of 13 exons, which span 7066 bp genomic sequences. Interestingly, the Arabidopsis PSD encoding region is also comprised of 13 exons, and their distribution� pattern in the genome is almost the same, suggesting the mechanism of PSD gene transcription and mRNA splicing among plants are much alike.

PSD homologs are conserved in evolution

Similar to other orthologs, the N-terminal of rice PSD contains xpo1 domain. The N-terminal is reportedly involved� in Ran-GTPase binding and interaction with leucine�-rich nuclear export signal sequences, whereas the C-terminal has been assumed to be a tRNA interaction region� that contributes to tRNA export [Fig. 1(B)] [19].

To better understand the position of rice PSD in the los1p/exportin-t gene family, we searched for proteins with an identity obviously similar to rice PSD in the NCBI protein� database, and we then sketched a phylogenetic tree. As expected, rice PSD is most identical to Arabidopsis PSD and the degree of similarity depends on the distance between� the species [Fig. 1(C)]. Together with other homologs� from human, mice and so on, they form a clade with high bootstrap support. Additionally, proteins from fungi form another independent clade. Reasonably, all these proteins come from eukaryotes, indicating that the origin of these homologs possibly occurred after the segregation of eukaryote and prokaryote.

Rice PSD was expressed throughout the development in normal plant

In order to study the general spatio-temporal expression pattern of PSD in normal rice plants, we subsequently examined the expression of PSD in various tissues from different phases of wild-type rice by RT-PCR. As expected, PSD was expressed throughout the development process [Fig. 2(A)], which is consistent with the fundamental nature� of its possible role in tRNA export.

Subcellular localization of the rice PSD protein in onion epidermal cells

Transient expression of GFP gene fusion in onion epidermis� is a credible method for the investigating subcellular localization� of GFP protein in plant cells [20]. We used this method to study the subcellular localization of rice PSD, and results showed that GFP and GFP-PSD were both distributed throughout the cells [Fig. 2(B)], implying that PSD shuttle between nucleus and cytoplasm to perform� its function.

Knockout of rice putative PSD transcript by T-DNA insertion caused severe development defects

To further investigate rice PSD function, we obtained T2 generation T-DNA insertional mutant line (1B-01410) seeds from Plant Functional Genomics Laboratory to insert within the gene [Fig. 3(A)] [10,11]. Thirty-one homozygote plants were chosen with common PCR reaction. The wild-type and heterozygote plants showed a sharp band (409 bp) with genomic forward and reverse primers designed around the insertion site, while the homozygote plant exhibited� a negative result. Another pair of T-DNA primers� was used to differentiate heterozygote from wild-type plants; 19 heterozygote plants showed an obvious PCR product of 543 bp while none of wild type did [Fig. 3(B)]. To confirm the knockout of PSD transcript in homozygote, we examined the PSD expression in leaf tissue with RT-PCR, and results revealed that T-DNA insertion completely disrupted the PSD expression [Fig. 3(C)].

Nearly all the homozygote plants displayed development retardation, such as delayed panicle heading and reduced size in the first several development stages, compared with the wild type [Fig. 3(D), upper]. These results are not so obvious for the Arabidopsis psd mutant possibly because of the different mutant pattern or the deviation of plant species. When the plants grew mature, their height and appearance were almost indiscernible [Fig. 3(D), bottom]. However, for plants losing PSD function, the seed-setting rate decreased significantly [Fig. 3(E,F)], as was nearly the case for Arabidopsis psd mutant [21]. Taken together, loss of function of PSD severely impaired the normal rice development process, and the most notable sharing phenotype� with Arabidopsis psd mutant implies that the functions of the homologs are coupled.

Rice PSD might regulate the tRNA export in vivo

To examine the role of rice PSD in tRNA export, we examined� the distribution of intronless tRNA-Tyr and intron�-containing pre-tRNA-Ala (GGC) in the nuclear and cytoplasmic� fractions of leaf tissue from wild-type and psd double mutant with Northern blot analysis. As anticipated, the mutant slightly decreased the level of both tRNA in the cytoplasm and increased their levels in the nucleus. We were particularly interested in the mutaton�s effect on pre-tRNA-Ala because accumulation of unspliced tRNA can be used to diagnose defects in tRNA export in yeast. Moreover, as the Arabidopsis psd mutant increased the level of unspliced tRNA-Tyr [22], the much higher level of pre-tRNA-Ala in the nucleus of rice psd should provide evidence of its regulatory role in tRNA export in vivo (Fig. 4).

Discussion

PSD is a member of the importin b family of nucleo�cytoplasmic transport receptors. It is hard to predict the role for this superfamily. For example, exportin 5, the mammalian� ortholog of HASTY (HST) and another member� of importin b family, exports pre-microRNA, tRNA, a viral� hairpin RNA and proteins associated with these and other double-stranded RNA, while HST�s yeast ortholog, Msn5p, exports several different types of phosphorylated proteins and imports replication protein A [23]. Though rice PSD shares 51% sequence identity with Arabidopsis PSD, the experimental validation is much more persuasive and necessary� to support the idea that they share the same function. The ever-growing populations of T-DNA insertions� in rice represent such a powerful tool for study of gene function. In this study, we verified a rice T-DNA insertional mutant psd and substantiated the function assignment� of rice PSD. The phenotypes of rice psd mutant� were not investigated in detail and other useful clues may have been missed. These phenotypes are possibly caused by the loss of function of rice PSD. An efficient way to confirm this point would be to construct the over-expressed� RNA interference vector to perform the complementation� test, and then observe the phenotypes of the transgenic plants. Considering that rice PSD is the homolog of Arabidopsis PSD that provided our bio�chemical data about the mutant, we assumed that these phenotypes likely originated from the impairment of tRNA export and the resultant translation limitation, which might elucidate rice PSD function to some extent.

In Arabidopsis, PSD transcript was observed in a series� of tissues such as roots, vegetative leaves, floral buds, shoot apex and so on. Its omnipresence was also concluded� in rice thus we did not quantitatively measure the expression� level. Similar omnipresence was also concluded in Arabidopsis. RT-PCR application merely amplified part of the PSD transcript, so we could not exclude the possibility� that PSD was differentially spliced in some tissue regions that we assayed. We also could not rule out the transcriptional regulation of PSD at a tissue- or cell-specific level. Further study of PSD regulation should increase our understanding� of its role in tRNA export pathways and its function in rice development.

Inhibition of tRNA nuclear exporter in yeast and Arabidopsis caused the accumulation of intron-containing pre-tRNA, leading to the prediction that pre-tRNA splicing could occur in the cytoplasm [22], and the case is the same for rice. Based on the expression and characterization� of rice PSD, we proposed a simple hypothetical model for the involvement of PSD in the process of tRNA nuclear export. Intronless and intron-containing pre-tRNA were transcribed, processed, edited in the nucleus and then exported� to the cytoplasm in the presence of PSD and other associated factors through the nuclear pore complex. In the cytoplasm, the tRNA cargo was released for later splicing (for pre-tRNA), translation or aminoacylation.

If rice PSD indeed functions as tRNA exporter in vivo, we speculated that it might not be the only tRNA export receptor in rice; though there were no other homologs according to BLAST up-to-date rice genome database. Also, notably, null allele of this gene was viable. Actually, Arabidopsis psd mutant slightly increased the level of intronless tRNA-Met in the nucleus, but it did not affect the cytoplasmic level of this tRNA [6], which demonstrated that it is not absolutely required for this process. Based on sequence alignment and conservation analysis, we tentatively� annotated the function domain of rice PSD. However, it was rather difficult to predict the in vivo interaction� mechanism due to a lack of a consistent in vitro assay to recreate events that occur during tRNA generation, processing and RNA-protein assembly, especially� in plants [24]. Moreover, we do not have a profound� structural knowledge of tRNA export. Deciphering� the complicated interaction between PSD with tRNA is therefore still a major challenge [25-27].

References

 1�� Kohler A, Hurt E. Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 2007, 8: 761-773

 2�� Rodriguez MS, Dargemont C, Stutz F. Nuclear export of RNA. Biol Cell 2004, 96: 639-655

 3�� Arts GJ, Kuersten S, Romby P, Ehresmann B, Mattaj IW. The role of exportin-t in selective nuclear export of mature tRNAs. EMBO J 1998, 17: 7430-7441

 4�� Li J, Chen X. PAUSED, a putative exportin-t, acts pleiotropically in Arabidopsis development but is dispensable for viability. Plant Physiol 2003, 132: 1913-1924

 5�� Lipowsky G, Bischoff FR, Izaurralde E, Kutay U, Schafer S, Gross HJ, Beier H et al. Coordination of tRNA nuclear export with processing of tRNA. RNA 1999, 5: 539-549

 6�� Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS. Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A 2005, 102: 3691-3696

 7�� Simos G, Hurt E. Transfer RNA biogenesis: a visa to leave the nucleus. Curr Biol 1999, 9: 238-241

 8�� An S, Park S, Jeong DH, Lee DY, Kang HG, Yu JH, Hur J et al. Generation and analysis of end sequence database for T-DNA tagging lines in rice. Plant Physiol 2003, 133: 2040-2047

 9�� Jeon JS, Lee S, Jung KH, Jun SH, Jeong DH, Lee J, Kim C et al. T-DNA insertional mutagenesis for functional genomics in rice. Plant J 2000, 22: 561-570

10� Jeong DH, An S, Kang HG, Moon S, Han JJ, Park S, Lee HS et al. T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 2002, 130: 1636-1644

11� Jeong DH, An S, Park S, Kang HG, Park GG, Kim SR, Sim J et al. Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J 2006, 45: 123-132

12� Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 15: 4876-4882

13� Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999, 41: 95-98

14� Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 2002, 18: 502-504

15� Richards E, Reichardt M, Rogers S. Preparation of genomic DNA from plant tissue. Curr Protoc Mol Biol 2001, 5: 2-3

16� Pawlowski K. Kunze R. Vries S, Bisseling T. Isolation of total, poly(A) and polysomal RNA from plant tissues. Plant Mol Biol Manual 1994, D5: 11-13

17� Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, Hartmann E, G�rlich D. Identification of a tRNA-specific nuclear export receptor. Mol Cell 1998, 1: 359-369

18� Arts GJ, Fornerod M, Mattaj IW. Identification of a nuclear export receptor for tRNA. Curr Biol 1998, 8: 305-314

19� Kuersten S, Arts GJ, Walther TC, Englmeier L, Mattaj IW. Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains. Mol Cell Biol 2002, 22: 5708-5720

20� Hanson MR, K�hler RH. GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 2001, 52: 529-539

21� Hunter CA, Aukerman MJ, Sun H, Fokina M, Poethig RS. PAUSED encodes the Arabidopsis exportin-t ortholog. Plant Physiol 2003, 132: 2135-2143

22� Hopper AK, Shaheen HH. A decade of surprises for tRNA nuclear-cytoplasmic dynamics. Trends Cell Biol 2008, 18: 98-104

23� Shibata S, Sasaki M, Miki T, Shimamoto A, Furuichi Y, Katahira J, Yoneda Y. Exportin-5 orthologues are functionally divergent among species. Nucleic Acids Res 2006, 34: 4711-4721

24� Yao C, Zhao B, Li W, Li Y, Qin W, Huang B, Jin Y. Cloning of novel repeat-associated small RNAs derived from hairpin precursors in Oryza sativa. Acta Biochim Biophys Sin 2007, 39: 829�834

25� McGuire AT, Mangroo D. Cex1p is a novel cytoplasmic component of the Saccharomyces cerevisiae nuclear tRNA export machinery. EMBO J 2007, 26: 288-300

26� Ghavidel A, Kislinger T, Pogoutse O, Sopko R, Jurisica I, Emili A. Impaired tRNA nuclear export links DNA damage and cell-cycle checkpoint. Cell 2007, 131: 915-926

27� Hutten S, Kehlenbach RH. CRM1-mediated nuclear export: to the pore and beyond. Trends Cell Biol 2007, 17: 193-201