Molecular cloning and characterization of rat karyopherin α1 gene: structure and expression

Gene 331 (2004) 149 – 157 www.elsevier.com/locate/gene

Molecular cloning and characterization of rat karyopherin a1 gene: structure and expression Bingwei Wang a,b,1, Zhihua Li a,1, Lei Xu a, Julian Goggi c, Yi Yu a, Jiawei Zhou a,* a

Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China b College of Animal Medicine, Northeast Agricultural University, Harbin 150030, China c MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK Received 3 December 2003; received in revised form 27 January 2004; accepted 4 February 2004 Reviewed by T. Gojobori

Abstract Dopamine denervation in the striata of patients with Parkinson’s disease (PD) leads to changes in neural plasticity. However, the mechanisms leading to the changes are still poorly understood. In an effort to study the molecular events in the denervated striatum, we identified and cloned rat karyopherin a1 (KPNA1), a member of the importin/karyopherin a (KPNA) family. DNA sequence analysis revealed that the full-length cDNA, encoding rat KPNA1, was 4975 bp with a short 5V-untranslated region (UTR) of 70 bp, a putative coding sequence of 1617 bp, and an unusually long 3V-UTR of 3266 bp. The gene shared a high degree of similarity with its mouse and human homologs at both cDNA and protein levels. By computational analysis of its genomic sequence, the transcription unit was shown to span a 44-kb region and consist of 13 exons varying in size from 89 (6th exon) to 3454 bp (13th exon), and 12 introns varying in size from 0.3 to 8.9 kb. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that KPNA1 transcript existed in various adult tissues. Both Northern blot and semi-quantitative RT-PCR analysis showed that the expression level of KPNA1 mRNA was altered in the denervated striatum post-lesion in a time-dependent manner, reaching the maximum at 2 weeks post-lesion. Our results suggest involvement of KPNA1 in the striatal responses to denervation following 6-hydroxydopamine (6-OHDA)-induced lesion. D 2004 Elsevier B.V. All rights reserved. Keywords: 6-Hydroxydopamine; Brain injury; Importin a; Karyopherin a

1. Introduction Parkinson’s disease (PD) is a very common neurological disorder affecting more than 2% of the population over 65 years of age. The most striking neuropathological features of PD include depigmentation of the substantia nigra (which results from a loss of pigmented dopaminergic neurons) and the presence of Lewy bodies in surviving neurons. The

Abbreviations: KPNA, karyopherin a; NLS, nuclear localization signal; NPC, nuclear pore complexes; 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; RACE, rapid amplification of cDNA ends; STAT, signal transducers and activators of transcription; UTR, untranslated region. * Corresponding author. Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Building 23, Room 316, 320 Yueyang Road, Shanghai 200031, China. Tel./fax: +86-21-5492-1073. E-mail address: [email protected] (J. Zhou). 1 These authors contribute equally to this work. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.02.009

severe degeneration of substantia nigra neurons results in the denervation of dopaminergic terminals in the nucleus striatum, the main recipient of nigral fibers. This denervation initiates a complex molecular process in striatal neurons and/ or glial cells to limit the extent of damage and reduce further injury. Previous studies have demonstrated that expression levels of several neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and members of the glial cell line-derived neurotrophic factor (GDNF) family, were elevated in the denervated striatum (Carvey et al., 1991; Zhou et al., 2000). Furthermore, clinical evidence has reported that striatal extracts from patients with PD are able to promote dopaminergic neuron growth in fetal mesencephalic cultures (Carvey et al., 1993). However, the increased synthesis of trophic factors in the target region after denervation is only part of the brain’s response to injury. The loss of dopaminergic inputs to the striatum also results in the profound reorganization of neuronal activity via specific

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dopamine-regulated cellular functions (Calabresi et al., 2000). However, the signaling cascades leading to these changes remain largely unknown. Understanding the modulation of cellular signaling in the striatum of an animal model of Parkinson’s disease is an important step in unraveling the pathogenesis of Parkinson’s disease and in developing novel approaches to repair injured or diseased brain. The final step of most intracellular signaling cascades, in response to environmental stimuli, is the nuclear transport of various regulators, including transcription factors, to modulate expression level of effecter genes or secondary signaling proteins. These regulators enter the cell nucleus through nuclear pore complexes (NPCs). The NPC consists of diffusion channels that permit the passive diffusion of small molecules, such as ions and proteins ( < 20 to 40 kDa). For larger proteins (>40 kDa), trafficking between the nucleus and the cytoplasm is mediated by a series of transport proteins. Several of these soluble transport proteins have been identified so far, including the small GTPase Ran/TC4, importin a, importin h, and others (Komeili and O’Shea, 2000). Importin a, also referred to as karyopherin a, is an adapter molecule. It binds to nuclear localization signal (NLS)-bearing proteins via two NLS-binding sites in the central area. Importin a also binds to importin h via its amino-terminal importin h binding (IBB) domain. Importin h transports the importin a/NLS complex to the nuclear side of the NPC. Once inside the nucleus, importin h binds to RanGTP (generated within the nucleus by the chromatinbound RanGDP/GTP exchange factor RCC1) leading to the dissociation of the import complex (Gorlich et al., 1996). Subsequently, importin h is thought to return to the cytoplasm independently of other export factors, whereas the export of importin a relies on the export factor CAS, which binds to importin a preferentially in the presence of RanGTP (Kutay et al., 1997). In any given organism, importin h is represented by a single gene. In contrast, several isoforms of importin a exist that form a protein family. In humans, the family consists of six members, including importin a1/karyopherin a2 (KPNA2) (Cuomo et al., 1994), importin a3/karyopherin a4 (KPNA4) (Seki et al., 1997), importin a4/karyopherin a3 (KPNA3) (Nachury et al., 1998), importin a5/karyopherin a1 (KPNA1) (Cortes et al., 1994), importin a6/karyopherin a5 (KPNA5) (Kohler et al., 1997), and importin a7/karyopherin a6 (KPNA6) (Kohler et al., 1999). In rat, only one member of the family has been identified, namely karyopherin a2 (Guillemain et al., 2002). As different importin a proteins differ in their substrate-specific import efficiency (Kohler et al., 1999), it is of interest to know whether other members of the importin/karyopherin family also exist in rat, one of the most commonly used model animals. Here, we have reported the cloning of the complete sequence of rat KPNA1 and the computational analysis of its genomic structure. We have also shown that expression levels of rat KPNA1 mRNA were increased in a timedependent manner in the dopamine-denervated striatum of

a rat model of PD. The possible mechanisms of its upregulation and the role KPNA1 might play in the central nervous system have been discussed.

2. Materials and methods 2.1. Cloning of cDNA encoding rat KPNA1 using 3V- and 5V-rapid amplification of cDNA ends (RACE) The Marathon-Ready rat brain cDNA library (Clontech, Palto Alto, CA, USA) was used to amplify the cDNA ends of the rat KPNA gene. The 3V-RACE gene-specific primer (GSP) used was 5V-TGT AGA CTG CCC TCA CTC GCC CAC T-3V (25 n.t., GSP3), and the 5V-RACE GSP was 5VTGG CAA GAA CAA GTG TCC ACA GAC GAG-3V (27 n.t., GSP5). Two and a half microliters of the cDNA template was used in the amplification. Cycle conditions were determined according to the manufacturer’s instructions. The resulting products were subcloned into pGEM T-easy vector (Promega, Madison, WI, USA) and sequenced. Further amplifications with RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed specific for the consensus sequence closing to the 5V- and 3V-end were performed to ensure that the complete sequence of the transcript was cloned. 2.2. Computational sequence analysis and determination of exon– intron boundaries The putative amino acid sequence of the obtained rat KPNA1 was aligned using the Clustal W Multiple alignment program, version 1.7 (Thompson et al., 1994), with several species’ KPNA1 protein sequences taken from the GenBank (http://www.ncbi.nlm.nih.gov). The cDNA sequence was blasted against the rat genome sequence database in the GenBank and the exon – intron boundaries were inspected by eye. The genomic sequence of the gene obtained was submitted to Genscan (Burge and Karlin, 1997) at BioSino (http://www.biosino.org) to verify the positions of exon –intron boundaries. 3V-Untranslated region (UTR) analysis was performed at UTR Home Page (http://www.bighost.area.ba.cnr.it/BIG/UTRHome). 2.3. 6-Hydroxydopamine (6-OHDA)-induced lesions and behavioral tests All animal experiments were carried out in accordance with the NIH guidelines. Female Sprague – Dawley rats weighing 180 – 220 g (provided by the Animal House, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were anaesthetized with Equithesin. Unilateral DA degeneration of the striatum was achieved by stereotaxic injections of 6-OHDA into the ascending medial forebrain bundle as described previously (Ungerstedt and Arbuthnott, 1970). Briefly, 4 Al of 6-OHDA (2 Ag/Al in 0.2

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mg/ml ascorbate – saline) was injected into the target area at the following coordinates: 4.4 mm caudal to bregma, 1.2 mm lateral to midline, 7.8 mm below dura. Sham-lesioned animals received 4 Al of the ascorbate vehicle. The lesion parameters used in the present study resulted in the selective destruction of virtually all dopaminergic neurons in the substantial nigra (>95%) and the ventral tegmental area (>80%), as was indicated by reduction in mRNA levels of tyrosine hydroxylase (TH), DA content and TH immunoreactivity. One or two weeks after lesioning, rotational behavior was assessed with apomorphine (0.05 mg/kg). The animals that exhibited adequate turning (i.e., at least six full body turns per minute contralateral to the lesion side) were used for further study. The brains were removed at 1, 2 or 5 weeks post-surgery and the striata ipsilateral to the lesion side were then dissected out and stored at 70 jC until use. 2.4. Semi-quantitative reverse transcriptase-polymerase chain reaction The animals were sacrificed by neck dislocation and various tissues of interest were removed quickly. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Single-strand cDNA was synthesized from 1 Ag total RNA in a volume of 20 Al containing 50 pmol random hexamers, 50 mM Tris –HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 50 mM DTT, 0.75 U Rnasin, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, and 200 U MMLV reverse transcriptase (Promega). Reactions were incubated for 1 h at 37 jC, terminated by heating for 5 min at 95 jC, and stored at 70 jC. Primers used to amplify a region of interest from rat KPNA1 were forward 5V-CCA GGA AAA GAG AAC TTT CGC CTG AAA-3V (P1) and backward 5V-CCT GCT CCT GGA CAT CTT CAA ACT CTG-3V (P2). Primers used to amplify a fragment from rat GAPDH were forward 5V-CCC ACG GCA AGT TCA ACG GCA3V and backward 5V-TGG CAG GTT TCT CCA GGC GGC-3V. Amplification was performed using Ex Taq system (Takara, Dalian, China). Briefly, after an initial denature at 95 jC for 3 min, the reaction mix was subjected to the following PCR conditions: 15 s at 94 jC, 30 s at 65 jC, and 1 min at 72 jC (26 cycles for KPNA1 and 22 cycles for GAPDH). The products were separated in 1.5% agarose gel and stained with ethidium bromide. To conduct isotope-labeled semi-quantitative PCR, 0.1 ACi 32P-labeled dATP was added to the reaction mix. The PCR conditions were the same as above except for the cycling numbers (22 cycles for KPNA1 and 18 cycles for GAPDH). The products were separated in 1.5% agarose gel and exposed to Storage Phosphor Screen (Dynamic Molecules) for 12 h. Negative control templates for each set of PCR reactions included RNA samples to show that no genomic DNA contaminant was introduced during reaction preparation. Negative controls also included H2O to demonstrate that no contaminants carry-over between samples.

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2.5. Northern blot analysis Total RNA was isolated from the DA-depleted striata of the rat 2 weeks after lesion or sham-operated animals. Thirty microgram of the total RNA was size-fractionated by 1.0% formalin-denatured agarose gel electrophoresis and transferred to a nylon membrane (Amersham Pharmacia Biotechnology, Uppsala, Sweden). A 556-bp cDNA probe specific for rat KPNA1 was PCR-amplified using primers P1 and P2 and was further 32P-labeled using a random primer labeling kit (Promega). After hybridization at 42 jC overnight, the blots were subjected to washing with ascending stringency. In order to check for total RNA quality and equal sample loading, blots were rehybridized with a 606bp rat GAPDH control probe. The blots were exposed to Kodak X-Omat K Film for 2 weeks (KPNA1) or for 12 h (GAPDH). 2.6. Statistical analysis Electrophoretic images and optical densities of amplified bands were analyzed using Scion Image (version B4.02). Statistical analysis was performed with commercially available statistical software (GraphPad Prism v4.0, GraphPad Software). The data were submitted to a one-way ANOVA followed by the Student – Newman– Keul’s test. Differences were considered significant at P < 0.05.

3. Results 3.1. Cloning of cDNA for rat KPNA1 To identify differentially expressed genes in the deafferented striatum in response to 6-OHDA lesions of the nigrostriatal pathway, PCR-based suppressional subtractive hybridization was employed. Over 60 genes or DNA fragments, whose expression levels were changed, have been identified including ribosomal protein L22, trophinin and cystatin C. Based on the sequence information of one of these fragments, primers for 3V- and 5V-RACE experiments were designed (GSP3 and GSP5, Fig. 1). Alignment and comparison of RACE fragments derived from rat brain revealed a consensus sequence containing all the characteristics of a transcript unit (translation initiation codon, translation stop codon, and polyadenylation signal). The full-length transcript was amplified with primers specific for the 5V- and 3V-end of the consensus sequence. The length of the cDNA we obtained was 4975 bp (including polyA tail), showing a short 5V-UTR of 70 bp, a putative coding sequence of 1617 bp, and an unusually long 3V-UTR of 3266 bp (GenBank accession no. AY351984). The coding region of the cDNA encoded a protein of 538-amino acid residues with a calculated molecular mass of 60,136 Da. The predicted protein product showed a high degree of homology with mouse and human importin a5/

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Fig. 1. The cDNA structure of rat KPNA1 gene. The bar represents the full-length cDNA, with the black part representing the open reading frame (ORF). The numbers below the bar indicate nucleotide number. The arrows indicate the relative positions of the translation initiation codon, the translation stop codon, and polyadenylational signal, from left to right, respectively. The curve at the end of the bar denotes a polyA tail. Arrowheads denote the relative positions of primers used in PCR amplifications. Note that the gene has a very long 3V-UTR, which is nearly two times as long as the ORF. On the contrary, the 5V-UTR is relatively short. The core hexamer sequence of the K box motif (TGTGAT) is located at nucleotides 2373 – 2378.

KPNA1 (Fig. 2). In fact, the sequence of the cDNA reported in the present study had 99% and 98% identities with mouse and human KPNA1 counterparts, respectively. The putative

ATG translation start codon was in a favorable context for translation initiation and no ATG codon could be detected upstream. A consensus polyadenylation signal (AATAAA)

Fig. 2. Comparison of amino acid sequences of KPNA1 from various species. Multiple alignments were conducted using the Clustal W program, version 1.7 (Thompson et al., 1994). Conserved amino acid residues are shaded in black. Amino acid residues are numbered at the top. R: rat; M: mouse (GenBank accession no. Q60960); H: human (GenBank accession no. P52294); Y: yeast (GenBank accession no. NP_014210).

B. Wang et al. / Gene 331 (2004) 149–157 Table 1 Genomic structure of the rat importin/karyopherin a1 subunit gene Exon

Position of the exon in cDNA (n.t. number)

5V-Donor site

Intron size (kb)

3V-Acceptor site

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

1 – 199 200 – 307 308 – 407 408 – 502 503 – 634 635 – 723 724 – 823 824 – 987 988 – 1066 1067 – 1192 1193 – 1320 1321 – 1499 1500 – 4953

GAACAGgtcggt GCACCAgtggta CAAAAGgtaagt CTGCAGgtgcgg GAGCAGgtaagg GCTGCAgtaagt GCTAAGgtaaat TCTGATgtaaga ACACAGgtatga ATCCAGgtaatg GATCAAgtaagc CATATGgtaagt

4.8 6.0 4.1 5.9 0.7 0.3 0.7 0.9 8.9 2.0 3.3 0.3

gttcagTTATTC gtccagGGGGGA aaacagAACATA ttgtagTTTGAA tcacagGCAGTC ttgtagGTTATT aaccagGTTTCT ttgtagGCACAA ttttagGTAATT tttcagACTGTG attcagTACCTA ttttagGTCTGG

Uppercase letters indicate exon sequences and lowercase letters indicate intron sequences. Note that all the introns conform to the GU – AG rule. n.t., nucleotide.

was found near the 3V-end of the cDNA (Fig. 1). These data indicate that we had cloned the full-length cDNA of rat KPNA1. 3.2. Computational analysis of the genomic structure and 3V-UTR of rat KPNA1 gene

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markedly uneven, ranging from 0.3 to 8.9 kb. To verify the exon – intron boundaries deduced, we submitted the sequence of NW_042728.1 to the Gensan server at BioSino (http://www.biosino.org). The predicted results obtained from the analysis showed that the rat KPNA1 gene contained 13 exons and 12 introns with exactly the same exon –intron boundaries as deduced from pairwise sequence comparison. Sequence comparisons of rat KPNA1 cDNA with the human and mouse counterparts in the database revealed that the rat KPNA1 we cloned had the longest 3V-UTR. At present, the explanation for this discrepancy is not known. It is likely that the existing mouse and human KPNA1 genes are partial sequences. Moreover, the analysis using UTRscan, an internet utility resource for functional UTR elements prediction, revealed the existence of a functional motif, called K box, in the 3V-UTR of both human, mouse and rat KPNA1. The location of K box in the 3V-UTR of rat KPNA1 was shown in Fig. 1. This motif, characterized by a core hexamer sequence (TGTGAT), was first identified in the 3V-UTR of Enhancer of Split Complex [E(spl) – C] genes and has been shown to be able to negatively regulate steady-state levels of E(spl) – C transcripts and proteins in the Drosophila (Lai et al., 1998). The presence of K box in the 3V-UTR of mammalian KPNA1 suggests that it may play a role in post-transcription regulation. 3.3. Expression profile of KPNA1 in the adult rat

To analyze the genomic structure of rat KPNA1 gene, the entire cDNA sequence obtained in this study was blasted against the rat genome sequence database in the GenBank. A Rattus norvegicus chromosome 11 WGS supercontig (GenBank accession no. NW_042728.1) was returned as the result and the exon – intron structure of the rat KPNA 1 gene was subsequently deduced using the pairwise alignment information. The gene spanned over 44-kb in the genome, including 13 exons and 12 introns. All the introns conformed to the GU –AG rule (Table 1). The lengths of the exons were around 100 bp, except for the last exon (3454 bp). The last exon results in an extraordinary long 3V-UTR, which may have a regulatory role in stability and translation efficiencies of the mRNA. The size distribution of the introns was

Expression of KPNA1 mRNA was determined using RTPCR in a variety of peripheral organs and tissues of adult rats. The primers were designed to amplify the region between the first and sixth exon. Since this region corresponding to the first six exons spanning over 35.1 kb in the genome, the possibility that the PCR product was generated from contaminating genomic DNA was excluded. To conduct semi-quantitative PCR, the concentrations of GAPDH cDNA of each sample was adjusted to the level as close as possible to each other before PCR amplification of KPNA1 was performed. As shown in Fig. 3, the KPNA1 mRNA was apparently abundant in the heart, kidney, placenta, pancreas, and spleen, whereas its expression

Fig. 3. Expression of KPNA1 mRNA in various tissues of adult rats revealed by RT-PCR analysis. KPNA1 was amplified for 26 cycles and GAPDH was amplified for 22 cycles. The KPNA mRNA was detectable in all rat tissues examined. Its expression levels appeared to be relatively higher in the pancreas, the spleen, and the placenta. Control RT-PCR with GAPDH transcript shows approximately equal loading of cDNA. H: heart; L: liver; S: spleen; K: kidney; B: total brain; M: skeletal muscle; Pa: pancreas; Pl: placenta; H2O lane represented a negative control without addition of the total RNA.

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levels in the brain, liver, and skeletal muscle were relatively lower. 3.4. Expression level of KPNA1 mRNA was elevated in the rat striatum following 6-hydroxydopamine-induced lesion To verify the upregulation of KPNA1 mRNA expression in the dopamine-depleted striatum following 6-OHDA-induced lesion of the ascending medial forebrain bundle, Northern blot analysis of the total RNA from the striatum of both 6-OHDA-lesioned and sham-lesioned rats was performed 2 weeks post-surgery. Consistent with the results in our RACE experiments, the rat KPNA gene was identified as a single transcript of approximately 4.9 kb as shown in Fig. 4. The mRNA level detected in the striatum of the lesion side appeared to be increased compared to that in control. Striping and reprobing of the same blots with probe of GAPDH indicated that the RNA of these samples were in good integrity and the absence of KPNA1 mRNA may be due to its relatively low abundance in the striatum of unlesioned side. Next, we investigated whether the increase of rat KPNA is in time-dependent fashion following the lesion. To carry out more precise quantitative analysis, we performed isotope-labeled RT-PCR. The animals received unilateral nigrostriatal lesion with 6-OHDA as described in Section 2.3 and the striata ipsilateral to the lesion side were then dissected out at 0, 1, 2, or 5 weeks after injury. It was observed that the expression level of KPNA1 mRNA in the 6-OHDA-lesioned striatum was elevated up to twofold at 2 weeks post-lesion compared with control ( P < 0.05, five independent experiments, Fig. 5B). Although it then declined at 5 weeks after surgery, it was still significantly higher than that in the control ( P < 0.05, Fig. 5A,B). These results suggested that 6-OHDA-induced lesion can change gene expression in the denervated target tissue, and that expression levels of KPNA1 mRNA in the striatum may be

Fig. 4. Differential expression of KPNA1 mRNA in the striatum following unilateral nigrostriatal lesion. The striata ipsilateral to lesion side were taken at 2 weeks post-lesion. Their total RNAs were extracted and subjected to Northern blot analysis (see Section 2.5). C: sham control. L: 6-OHDA lesioned. The positions of 28S and 18S rRNAs indicate approximately 4.9 and 1.9 kb, respectively.

Fig. 5. Differential expression of KPNA1 mRNA in the striatum ipsilateral to lesion side following 6-OHDA infusion. The animals received nontreatment (0 week) or administration of 6-hydroxydopamine at indicated time. The intensities of amplified DNA products increased through 18, 22, and 27 cycles (data not shown). (A) Representative photos of radiolabelled PCR products amplified with specific primer pairs for KPNA1 or GAPDH. (B) Quantitative results of (A). The optical densities of the bands were determined and the results were presented as ratio of optical densities of amplified band versus GAPDH (mean F S.E.M., n = 5). *P < 0.05, compared with normal control (0 week post-lesion), using one-way ANOVA followed by the Student – Newman – Keul’s test.

modulated by afferent dopaminergic input in a slow-rising and long-lasting fashion.

4. Discussion In the present study, we reported the molecular cloning and characterization of full-length cDNA encoding rat KPNA1, a member of the importin/karyopherin a family, and its characteristic gene structure and expression pattern. At least four names exist at present in the database for the human homologue of KPNA1 reported in the present study: SRP1, importin a5, karyopherin a1, and importin/karyopherin a1 subunit. In order to stay consistent with the nomenclature of the HUGO Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature), the gene reported here has been designated as rat importin a5/KPNA1. In humans, the importin/karyopherin a family can be divided into three sub-families according to the sequence homology. The first consists of importin a1 alone, the second of importins a3 and a4, and the third of importins a5, a6, and a7 (Kohler et al., 1999). Although several of these members have also been found in invertebrates, only one gene for importin a, SRP1, has been identified in the yeast Saccharomyces cerevisiae, which is the yeast ortholog of mammalian KPNA1 (Yano et al., 1992). The similarity

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between yeast SRP1 and rat KPNA1 is 65% at the amino acid level, and human KPNA1 shares up to 98% identity with rat KPNA1. The early origin of the SRP1 gene in evolutionary history and the high similarity among its orthologs in different species suggests that it may play an important role in the fundamental activity of cells. By computational analysis of the genomic sequence, it was found that the rat KPNA1 gene contained 13 exons spanning up to 44 kb in the genome. One significant feature of the rat KPNA1 transcript was that it contained an unusually long (3.3 kb) 3V-UTR arising from a single exon. The rat homolog of KPNA1 that we cloned had the longest 3V-UTR, which shared 92% or 86% homology with the same region of mouse or human KPNA1 cDNA. Among several mouse KPNA1 cDNAs deposited in the GenBank, the length of the longest 3V-UTR of mouse KPNA1 cDNA (GenBank accession no. NM_008465) is 2254 bp. As there is no polyA tail at the 3Vof the sequence, it is likely that this sequence, and perhaps others, only cover partial sequence of KPNA1 cDNA. On the other hand, another record in the GenBank (accession no. BC006771) indicates that mouse KPNA1 contains a polyA tail and a relatively short 3V-UTR at 914 bp, indicating the existence of alternative polyadenylation sites in mouse KPNA1. In contrast, the longest 3VUTR of human KPNA1 reported is 1277 bp (GenBank accession no. NM_002264), with no putative alternative polyadenylation site found. Although the 3V-UTRs of eukaryotic mRNAs were once thought to be unimportant trailers following protein coding regions, growing evidence now indicates that they in fact may influence polyadenylation efficiency, transcript localization, transcript stability, and translational efficiency (Chen and Shyu, 1995). The unusually long 3V-UTR of rat KPNA1 might be involved in the regulation of its expression level and/or function. This hypothesis is supported by the identification of a K box motif in the 3V-UTR of rat KPNA1. The K box motif, defined by a core hexamer sequence of TGTGAT, is an exact sequence identity present in one or more copies in the 3V-UTR of Brd and several genes of the E(spl) – C in Drosophila. It has been shown that the integrity of the K box sequence is essential for the strong, negative posttranscriptional regulation conferred on a heterologous lacZ reporter gene by a K box-containing 3V-UTR, furthermore, a lack of K box-mediated regulation has been shown to cause an attenuation of PNS defects in Drosophila (Lai et al., 1998). As the K box element was present in the 3V-UTR of both human, mouse and rat KPNA1, it is likely that the KPNA1 gene may have a similar role in gene regulation over various mammalian species. The exact role of K box in the regulation of KPNA1 expression following 6-OHDA lesioning will be addressed in future investigations. Fig. 3 indicates that although the expression of KPNA1 was detectable in various adult rat tissues, there was moderate difference in the abundance of the transcript between different tissues. Nachury et al. have shown that 4000-n.t.-long mRNA of hSRP1g could be detected in

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various tissues including skeletal muscle, pancreas, kidney, liver, lung, placenta, brain, or heart (Nachury et al., 1998). In agreement with their observations, we found that KPNA1 mRNA can also be detected in rat tissue. Previous studies have demonstrated that expression levels of importin a’s are regulated by various physiopathological conditions. Activation of human blood peripheral lymphocytes with lipopolysaccharide, concanavalin A, or combination of phorbol myristic acid and ionomycin causes significant increases in the expression levels of KPNA1 and KPNA2 in both the cytoplasm and nucleus (Nadler et al., 1997). In cultured Hela cells, serum depletion results in a reduction of all six importin a’s as well as importin h and CAS, whereas elevated expressions are observed 24 h after re-addition of serum. Importin a5/KPNA1 is also found to be transiently up-regulated in the promyelocytic cell line HL60 24 h after PMA-induced differentiation (Kohler et al., 2002). In two rat models of diabetic nephropathy, the expression level of importin a7 is increased significantly in the kidneys (Kohler et al., 2001). Given that importin a proteins are differentially regulated in these conditions, and they are reported to have substrate-specific import efficiencies (Nachury et al., 1998; Kohler et al., 1999), different importin/karyopherin a proteins may be involved in specific cellular function and may be also involved in diverse pathological processes. The elevated expression level of KPNA1 appears to be associated with neuronal differentiation. It has been reported that two rat ESTs (GenBank accession nos. H33259 and H33258), both corresponding to part of the 3V-UTR of rat KPNA1, are up-regulated 9 days after treatment of PC12 cells with nerve growth factor (NGF) (Lee et al., 1995). As NGF treatment of PC12 cells is a well-established in vitro model of neuronal differentiation, it suggests that KPNA1 may play a role in the development and/or repair processes of the nervous system. In agreement with this, we demonstrated in the present study that expression level of KPNA1 was up-regulated in the striatum of a rat model of PD, implicating the association of a member of importin/karyopherin a family with brain repair or neuroregeneration. Several proteins are known to be imported into the nucleus with the aid of KPNA1, including signal transducers and activators of transcription 1 and 2 (STAT1, 2), lymphoid enhancer factor-1 (LEF-1), and RAG-1 (Fagerlund et al., 2002). Of particular interest are STAT proteins, which have been shown to participate in various processes such as development of the central nervous system (DeFraja et al., 1998), ischemic brain injury, and regulating of expression of neuropeptides (Buzas et al., 1999). Type I interferon (IFN-a, -h, and -N) stimulation causes STAT1 and STAT2 to be phosphorylated and form heterodimers, while type II IFN (IFN-g) stimulation leads to phosphorylation and the formation of STAT1 homodimers. Both STAT1 – STAT2 heterodimers and STAT1 homodimers bind to KPNA1 and translocate into the nucleus to activate IFNresponsive genes (Fagerlund et al., 2002). Regulation of KPNA1 expression levels may be involved in the cellular

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mechanisms to control activation and nuclear translocation of STAT1 and STAT2. On the other hand, given that the import mediating factors for most nuclear proteins are still unknown and that substrate-specific import efficiencies of the different importin a’s have been reported earlier (Nachury et al., 1998; Kohler et al., 1999), there could be many other transcription factors and co-factors whose nuclear translocation are mediated and regulated by KPNA1. Furthermore, it has been suggested that importin a proteins have functions other than nuclear transport (Wen and Shatkin, 2000). Therefore, it is likely that KPNA1 is involved in multiple cellular regulatory mechanisms in response to external stimuli and its up-regulation contributes to a variety of cellular effects caused by dopamine denervation in the striatum, such as increased expression level of trophic factors and reorganization of glutamatergic transmission (Calabresi et al., 1993; Zhou et al., 2000). In conclusion, we have cloned the rat KPNA1 gene, a member of the importin/karyopherin a family, whose expression level was up-regulated in the rat striatum following 6-hydroxydopamine-induced lesion of the nigrostriatal pathway. Further investigations on the function of KPNA1 in this setting will help to further unravel molecular changes in the denervated striatum and pathogenesis of PD.

Acknowledgements This work was supported by grants from the Chinese Academy of Sciences (No. KSCX2-SW-209), the National Natural Science Foundation of China (No. 30000047 to L.X.), the National Basic Research Program of China (G1999054000), and the Chinese Ministry of Science and Technology (No. 2001AA221221).

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