Characterization on the alternative splicing

2008海峽兩岸大學校長論壇暨科學技術研討會

2008 年 5 月 26-27 日

Characterization on the alternative splicing, expression and gene phylogenesis of PTPR4 family in Japanese flounder, Paralichthys olivaceus Quanqi Zhang College of Marine Life Science, Ocean University of China Email: [email protected] PTPε M in zebrafish and described their expression during development. However, none of cytoplasmic form of PTPε was identified in teleost fish. Here we report the cDNAs of PTPα and two forms of PTPε, from Japanese flounder. We proved that these two isoforms of PTPε are also from one PTPε gene by alternative mRNA splicing. The unique expression of PTPα and two isoforms of PTPε mRNAs in various tissues are described.

ABSTRACT We have identified the PTP Receptor-Type IV (PTPR4) family, including one form of PTPα and two forms of PTPε (PTPε M and PTPε C) in flounder. The existence of PTPε C was the first report in non-mammalian animals. Semi-quantitative RT-PCR showed independent expression patterns of the three forms. The sequence of PTPε C was identical to that of PTPε M except for its 5'-terminal regions. Southern blot analysis proved that there existed only one PTPε gene in the genome, indicating that the two isoforms of PTPε might have been derived from alternative splicing of this gene. Phylogenetic analysis also provided evidence that the gene duplication from the ancestor gene to PTPα and PTPε occurred before the divergence of Gnathastomata and Agnatha. These results showed that the functional evolution of protein phosphorylation is promoted by not only genome duplication, but also elaborate regulation of expression.

MATERIALS AND METHODS Fish Sample The animal used was Japanese flounder, Paralichthys olivaceus collected from a flounder hatchery located in Weihai City. Isolation of PTPα and PTPε cDNAs RNAs were extracted individually from brain, spleen and kidney tissues using TRIZOL. The cDNAs were synthesized from RNAs by RT-PCR using oligo (dT) primer with reverse transcriptase MMLV (Promega). The sense primer for PTP (PTP-sense) and the antisense primer for PTPR4 (PTP-antisense) were designed from conserved PTP sequence (Denu et al., 1996) (Table 1).

INTRODUCTION Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs), is a critical mechanism for the regulation of numerous cell functions (Tonks and Neel, 1996; Mustelin et al., 2002). Since the first identification, PTP members have been identified in many organisms (Tonks et al., 1988; Fischer et al., 1991; Alonso et al., 2004). PTPR4 contain two cytoplasmic PTP domains (D1 and D2), one transmembrane segment and an extracellular domain. PTPα and PTPε are the only known members of PTPR4 (Sap et al., 1990; Matthews et al., 1990). The precise subcellular localization of PTPs is an important for regulating their physiological roles (Mauro and Dixon, 1994; Fischer, 1999). PTPε includes four forms of proteins coded by a single PTPε gene. The two most prevalent forms are the transmembrane form (PTPε M) and cytoplasmic form (PTPε C) derived from alternatively splicing of PTPε gene (Elson and Leder, 1995b; Elson et al., 1996). The other two forms p67 PTPε and p65 PTPε are produced by initiation of translation of PTPε mRNA, and specific proteolytic cleavage of larger PTPε proteins, respectively (Gil-Henn et al., 2001; Kraut et al., 2002). Each form possesses unique expression patterns, subcellular localizations, and functions. In teleost fish, Okubo and Aida (2003) isolated and characterized PTPα and PTPε M in medaka, and demonstrated that GnRH down regulates their expression. Van der Sar et al (2001) cloned PTPα and

Table 1

Primers used in this study

Name

Sequence

PTP-sence

5'-GAYTTCTGGMGRATGRTYTGG-3'

PTP-antisence

5'-YAGYTCYGTRTCNCCRTA-3'

PTPα-F1

5'-TGACCTCCTGGTCACCAACAACAG-3'

PTPα-F2

5'-GCATCCCCACTGACGGAAAAG-3'

PTPα-R1

5'-GGGACAGTCTGGAGCTCTGTATCATCCGA-3'

PTPα-R2

5'-TCGTCAGTCTGAAGGAATTTGAATG-3'

PTPε-F1

5'-GAGAAGCAGACGAGGGCGATCAGGC-3'

PTPε-F2

5'-TCCACGGCTGGCCGGAGATCG-3'

PTPε-R1

5'-GCGTCTTTGCGGCATCGCTGGCTTGGTA-3'

PTPε-R2

5'-ATGGTGTAATCCACCAGGACAGT-3'

PTPε-F3

5'-CAGACGGTGGTCCTTCTGCCTC-3'

PTPε-R3

5'-GCCGTCGTCAGCAGAGCGTAG-3'

PTPε-F4

5'-ACATCCCCCAGCCACCACAT-3''

PTPε-F5

5'-GAATAGATTCTCAAGTTTCAGATGG-3'

PTPε-R4

5'-CAGAGGTTTCCATCCAGCGG-3'

PTPα-F3

5'-ATCAAAGCCCCGTCACAAGTC-3'

β-actin-F

5’-GAGATGAAGCCCAGAGCAAGAG-3’

β-actin-R

5’-CAGCTGTGGTGGTGAAGGAGTAG-3’

The amplified fragments were cloned into PMD-18T vector (TaKaRa, China). Sequences from six clones were obtained and confirmed by multiple alignments of the deduced amino acid sequences using CLUSTAL W method (Thompson et al., 1994). The result suggested that three inserts each for PTPα and PTPε, respectively. 1


2008 年 5 月 26-27 日

Rapid amplification of cDNA ends (RACE) of PTPα and PTPε was carried out using SMART RACE amplification kit (BD Biosciences) with mixed RNA as templates. Gene specific primers (GSPs) PTPα-F1/PTPα-R1 and nested primers (NGSPs) PTPα-F2/PTPα-R2 were used for 3'- and 5'-RACE of PTPα, respectively. Likewise, GSPs PTPε-F1/PTPε-R1 and NGSPs PTPε-F2/PTPε-R2 were used for 3'- and 5'-RACE of PTPε, respectively (Table 1). Both 3'-RACE and 5'-RACE of reaction of PTPα produced only one band, but 5'-RACE reaction of PTPε generated two bands. All the products were cloned and at least three clones of each product were sequenced. Sequence Alignment Sequence data used for phylogenetic analysis were obtained from NCBI. Multiple alignment of the amino acid sequences of PTP domains D1 and D2 in PTPR4 were conducted using MEGA 3.1 software (Kumar et al., 2004). Southern blot analysis Genomic DNA was extracted individually from muscle tissues, digested completely with BamH I，EcoR I，Hind III, Pst I or Xba I, respectively. Samples were separated in 0.9% agarose gel, blotted to nylon membranes (Amersham) and cross linked under UV light. The hybridization probe, a 108 bp cDNA fragment hybridizing to a common sequence of PTPε cDNAs, was generated by PCR using primers PTPε-F3 and PTPε-R3 (Table 1), labeled with PCR DIG probe synthesis kit (Roche). After pre-hybridization at 44°C for 30 min, denatured probe was added to pre-heated DIG Easy Hyb at concentration of 1.5 μl/ml and hybridized to the membrane at 44°C for 12 hrs. The blot was washed to a final stringency of 0.5×SSC containing 0.1% SDS at 65°C and hybridization signal was visualized with NBT/BCIP (Roche). Semi-quantitative RT-PCR Analysis Aliquots of 2μg total RNAs from differen tissues were used to synthesize single-strand cDNAs. These cDNAs were diluted 10 folds with sterile water and used as templates for semi-quantitative RT-PCR. Primers PTPα-F3/PTPα-R2 were used for PTPα, PTPε M and PTPε-F4/PTPε-R4 for PTPΕε-F5/PTPε-R4 for PTPε C (Table 1). Duplex RT-PCR was performed to amplify PTPε M and PTPε C from the same cDNA samples using PTPε-F4, PTPε-F5 and PTPε-R4. Expression of β-actin served as internal control. The number of cycles was optimized to be 20 for the β-actin and 26-28 for PTP genes.

complete PTPα cDNA sequence of 3086 bp was obtained (Genbank, DQ683253). This sequence contained a 454 bp 5' UTR, a 2490 bp open reading frame (ORF) with a typical Kozak sequence and a 142 bp 3' UTR. The deduced protein of 830 amino acids included a probably signal peptide, transmembranal region and two highly conserved catalytic domains of receptor-type PTPs. The identity between flounder PTPα and human, mouse, rat, chicken, African clawed frog, zebrafish or medaka PTPα was 77%, 74%, 76%, 76%, 73%, 80% and 83%, respectively. The C-terminal, representing the cytoplasmic PTP domains and the transmembranal region, had high identity (91%, 90%, 90%, 89%, 91%, 96%, and 95%, respectively) but its N-terminal, the extracellular domain, showed very low identity (18%, 15%, 15%, 23%, 15%, 25%, and 43%, respectively) (Fig. 1). Structure of Two Isoforms of PTPε In 3'-RACE PCR for PTPε a single 1300 bp fragment including polyA tail was amplified. In 5'-RACE reaction, however, two different-sized PCR products were obtained. The longer one was 1100 bp and the shorter one was 850 bp. Their sequence difference located exclusively at the 5' ends. The first 392 bp of the larger cDNA fragment were replaced by 143 bp in the shorter one. Thus two complete cDNAs of PTPε were obtained with 2481 bp and 2232 bp (Genbank DQ825344 and DQ825345), respectively. Each cDNA contained an ORF and an in-frame stop codon upstream of the initiator ATG indicating that they were all the true complete cDNAs. The deduced amino acid sequences of the two proteins were identical except a small part at their amino end (Fig. 2A). The unique sequences of the larger PTPε consisting of 56 amino acid residues contained a signal sequence, an extracellular region, and a transmembranal domain. These regions were replaced in the smaller one by 11 hydrophilic amino acids (Fig. 2B). Both proteins consisted of two highly conserved catalytic domains of PTPs. Compared with human orthologs the larger one should be “PTPε-transmembranal” (PTPε M), while the shorter one “PTPε-cytoplasmic" (PTPε C) Flounder PTPε M shared 73%, 72%, 72%, 84% and 77% identity with human, mouse, rat, zebrafish and medaka PTPε M, respectively. The D1 and D2 domain of flounder PTPε exhibited 79%, 78%, 78%, 88% and 82% identity with the corresponding regions in human and other animals, respectively (Fig. 3). Flounder PTPε C shared 77% and 76% identity with human and rat PTPε C, respectively (Fig. 4). The Number of PTPε Genes in Flounder Genome To clarify how many PTPε genes exist in flounder genome, southern hybridization was carried out using probe designed to hybridize a common sequence of both isoforms (Fig. 2). The results showed only one hybridization signal in all BamH I，EcoR I，Hind III, Pst I or Xba I digested genomic DNA (Fig. 5).

RESULTS Isolation and Sequencing of PTPα. A 560 bp fragment was obtained by general primer for PTP and specific primer for PTPR4. Six independent clones were sequenced. Sequence analysis showed that three inserts were PTPα and the other three were PTPε. Based on this sequence, a 1230 bp fragment was obtained by 3'-RACE PCR of PTPα, and a 1680 bp fragment was amplified in 5'-RACE. Thus the 2


2008 年 5 月 26-27 日

Phylogenetic Analysis of PTPR4 For phylogenetic analysis of PTPR4 family among cyclostomes, cartilaginous fishes, teleosts and tetrapods, only sequences PTP domain 2 (D2) and part of D1 are available for all the taxa. The phylogenetic trees by both D1 and D2 showed that the PTPR4 was divided clearly into two branches, PTPα and PTPε. Flounder was first jointed with zebrafish or medaka, and then the three teleosts formed a cluster in both branches. In hagfish, no PTPε and PTPα D1 are available for phylogenetic analysis at present, the hagfish hgPTPR4 was apparently an ortholog of human / mouse / rat / chicken / African crawed frog PTPα(Fig. 6).

Tissues Expression of PTPR4 Semi-quantitative RT-PCR revealed that PTPα expressed highly in gill, and expressed at different levels in brain, heart, spleen, kidney, intestine and testis. The expression in liver, muscle and ovary tissues was very low or undetectable (Fig. 7). PTPε M expressed in all tissues, brain tissue showed the highest expression level and liver showed the lowest. In comparison, the expression of PTPε C showed higher variation among tissues. It expressed relatively high in spleen and kidney tissues, but very low or no expression other tissues (Fig. 7). There were no positive correlations in the expression patterns as well as the expression levels between these two forms of PTPε (Fig. 7).

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2008 年 5 月 26-27 日

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PTPε M only at its 5' end. The identification of PTPε C in non-mammalian animals has not been reported yet. In mammals, the two isoforms of PTPε are encoded by a single gene (Elson et al., 1996; Tanuma et al., 1999). To explore the genome organization of PTPε, genomic Southern blot analysis was carried out using probe designed to hybridize a common sequence of both isoforms. Only one hybridization signal was detected in each digested DNA. This fact proved that there exists only one PTPε gene in flounder. The two cDNA isoforms might have been derived from alternative splicing of the same PTPε gene as in mammalian genome (Elson et al., 1996; Tanuma et al., 1999). Each isoform of PTPε possessed unique tissue-specific expression patterns in flounder, indicating that which isoforms is to be expressed might be subject to complicated regulation at the level of transcription. The distinct expression levels and patterns in various tissues suggested that each isoform should have distinct physiological roles. Studies in many eukaryotic gene families have proved that the multiplicity of members in the same family rapidly increased in the first half of chordate evolution before the fish–tetrapod split by gene duplications (Iwabe et al., 1996). The phylogenetic position of the hagfish hgPTPR4 in the phylogenetic tree by PTP domain D2 apparently showed that it is an ortholog of PTPα. At present PTPε has not been isolated from hagfish, it is thus difficult to say whether there exists PTPε in this animal. PTPεfrom other species in this phylogenetic form a cluster first and then joined with the above PTPα branch. This fact indicated that PTPα and PTPε were from a common ancestor. Judging from the phylogenetic position of hgPTPR4 by PTP domain D2 of PTPR4, the duplication from the ancestor gene to PTPα and PTPε was likely before the divergence of Gnathostoma and Agnatha. To our limited knowledge, human PTPε is the first phosphatase reported to express both isoforms. It is interesting that teleost fish,

DISCUSSION Here we have isolated the full length cDNAs of PTPR4 family, including one form of PTPα and two forms of PTPε (PTPε C and PTPε M) from Japanese flounder. Sequefce of PTPε C differed from that of 5


2008 年 5 月 26-27 日

the evolutionarily lower Gnathostoma, had developed the function of alternative expression just after genome duplication.

ACKNOWLEDGEMENTS This work was supported by grants from the National 863 Progrma (No. 2006AA10A404).

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