Gene 267 (2001) 89±93www.elsevier.com/locate/gene Identi®cation of alternative splicing of spinocerebellar ataxia type 2 gene Adelina Affaitati 1, Tiziana de Cristofaro 1, Antonio Feliciello, Stelio Varrone* Centro di Endocrinologia ed Oncologia Sperimentale (C.E.O.S.) del C.N.R., c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita di Napoli `Federico II', Via Pansini 5, I-80131 Napoli, Italy Received 12 October 2000; received in revised form 12 February 2001; accepted 19 February 2001 Received by R. Di Lauro Abstract Spinocerebellar ataxia 2 (SCA-2) is a neurodegenerative disorder caused by the expansion of an unstable CAG/polyglutamine repeat located at the NH2-terminus of ataxin-2 protein. Ataxin-2 is composed by 1312 aminoacids and it is expressed ubiquitously in human tissues. To date, the function of ataxin-2 is not known. In this study, we report the characterization of an alternative splice variant of human ataxin-2. The splice transcript lacks the exon 21 and connects exon 20 to exon 22 with the same reading frame of the full length mRNA. This novel isoform of ataxin-2 is conserved in the mouse. It is named type IV to differentiate it from type II splice variant lacking exon 10 (present in human and mouse cDNAs) and from type III, lacking exon 10 and exon 11 seen in mouse. Type IV of human ataxin-2 cDNA is predicted to encode a protein of 1294 residues. Both the full length and the type IV transcript of ataxin-2 are present in several human tissues, including brain, spinal cord, cerebellum, heart and placenta. These ®ndings allow the hypothesis that type I, II and IV of human ataxin-2 might perform different functions. q 2001 Published by Elsevier Science B.V. All rights reserved. Keywords: CAG repeat; Neurodegenerative disorders; Ataxin-2 isoforms; Genomic structure; bp, base pair (s); cDNA, DNA complementary to RNA; dNTP, deoxyribonucleoside triphosphate; kb, 1000 bp; kDa, Kilodalton (s) 1. Introduction The polyglutamine neurodegenerative diseases represent a group of disorders that include spinobulbar muscular atrophy (SBMA) (La Spada et al., 1991; Nakamura et al., 1994), Huntington's disease (HD) (The Huntington's Disease Collaborative Research Group, 1993), dentatorubropallidoluysian atrophy (DRPLA) (Nagafuchi et al., 1994; Koide et al., 1994) and the spinocerebellar ataxias (SCAs) (Ban® et al., 1994; Orr et al., 1993; Kawaguchi et al., 1994; David et al., 1997; Zhuchenko et al., 1997; Riess et al., 1997). The molecular basis of polyglutamine diseases is a dominant toxic gain of function that occurs at protein level characterized by the presence of about 35±40 residues of glutamine, due to unstable expansion of CAG trinucleotide repeats in the coding region of the gene (Housman, 1995). Polyglutamine expansion could cause mutant proteins to adopt altered conformation, leading to their ubiquitination, aggregation Abbreviations: mRNA, messenger RNA; .PCR, polymerase chain reaction; poly(A) 1, polyadenilated RNA; RT-PCR, reverse transcriptase-polymerase chain reaction * Corresponding author. Fax: 139-081-770-1016. E-mail address:
[email protected] (S. Varrone). 1 Adelina Affaitati and Tiziana de Cristofaro contributed equally to the manuscript. and cell death. Intranuclear inclusions composed of aggregated proteins appear to be a common feature in the pathology of the CAG repeat (Li and Li, 1998). We have recently demonstrated that the length of polyglutamine, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are critical for the accumulation of intracellular aggregates (de Cristofaro et al., 1999). Moreover, in the recent work we have showed that, in human neuroblastoma cells, nuclear inclusions induced by polyglutamine-expanded protein result in cell death (de Cristofaro et al., 2000). Spinocerebellar ataxia 2 (SCA-2) is an autosomal dominant disorder leading to neuronal degeneration, primarily in the cerebellum, but also in other parts of the central nervous system (Pulst et al., 1996; Imbert et al., 1996). Epidemiologic studies indicate that SCA-2 is particularly present in southern Italy and in north Europe (Filla et al., 1999). The gene causing SCA-2 has been recently isolated and named ataxin-2. Expansion of CAG/polyglutamine tract, located at NH2 terminus of ataxin-2, is linked to the accumulation of intracellular inclusions and neuronal cell death (Koyano et al., 1999). Ataxin-2 gene is constituted by 25 exons; northern analysis identi®ed a major transcript of 4.5 kb and the open reading frame consists of 3936 bp. Ataxin-2 is composed by 0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00402-4 The transcript is predicted to encode a novel isoform of ataxin-2 widely expressed in human tissues. while type II and type III lack exon 10. 1989). 2 ml of mouse cDNA were added to 50 ml of the PCR mix containing 200 mM of dNTPs and 20 mM of sense primer and antisense primer corresponding to nucleotide positions 1242±1267 (5 0 GGGAAGCAAGGGCAA ACCAGTTAGCA-3 0 ) and 3948±3925 (5 0 -TCCGGTTGGCAGAAGCAGAGAA G 3 0 ) of mouse ataxin-2 cDNA (GenBank Accession No.CTGGCTTTGCTGCTGTCCGGTGG-3 0 ) of mouse ataxin-2 cDNA. Sequence analysis con®rmed the identity of ampli®ed cDNA with ataxin-2 sequence. 10 ml of dT oligo were used both on 1 mg of total RNA and 0. two fragments of different size (267 . 1989) was isolated from peripheral blood lymphocytes by using the TRIZOL Reagent Kit (Life Technologies). Furthermore. Ampli®ed products were resolved on 2% agarose-gel. In the present work. heart and placent was purchased from Clontech. Results and discussion To isolate full length ataxin-2 cDNA. puri®ed by using gel extraction kit (QIAGEN). 2. last cycle was extended for 5 min at 688C. In central nervous system. PCR reaction mixture was resolved on 1% agarosegel and visualized with 10 mg/ml ethidium bromide. Mouse cDNA from brain. 30 cycles were repeated as follows: denaturation at 948C for 10 s. while the splice variant is more abundant in cerebellum (Sahba et al. Reverse transcribed cDNA was used as template for a polymerase chain reaction with oligonucleotide primers spanning 1469±4165 bp of SCA-2 gene. respectively (Nechiporuk et al.. with conserved reading frame downstream the splicing site. As shown in Fig. by using CEQ 2000 DNA Analysis System. spinal cord. we employed polymerase chain reaction on reverse transcribed total RNA isolated from human peripheral blood lymphocytes with two primers located at the 5 0 and 3 0 of the published sequence (see Section 2). we tested the presence of this variant transcript in the cDNA derived from several tissues. 1998).. exten- sion at 688C for 1 min. three isoforms of mouse homolog of SCA-2 gene have been reported: type I contains the full-length cDNA. cerebellum. 1. we report a new splice variant of human SCA-2 gene that lacks the exon 21. Reverse transcriptase-polymerase chain reaction (RTPCR) Total RNA of control or SCA-2 patients (Sambrook et al. Sequences generated using Sp6 and T7 primers were compared to human and mouse ataxin-2 cDNA sequence and the splice junctions were determined at the point of divergence between both sequences. Total RNA was extracted on mouse embryonic days 12±17 (E12±E17) by using the TRIZOL Reagent Kit (Life Technologies). Sp6 and T7 oligonucleotide primers were used. respectively.. PCR product was then subjected to second PCR by using internal oligonucleotides primers spanning 3372±3639 bp of full-length SCA-2 cDNA. CA).2. 2. PCR conditions 2 ml of the ®rst-strand mixture were added to 50 ml of the PCR mix containing 200 mM of dNTPs and 20 mM of sense primer and antisense primer corresponding to nucleotide positions 1469±1494 (5 0 -GGGAAGCAAGGGCAAACCAGTTAGCA-3 0 ) and 4165±4142 (5 0 -TCCAGTTGGTAGAAGCAGTAGAAG-3 0 ) of human ataxin-2 cDNA (GenBank Accession No. or exons 10 and 11. AF041472). Materials and methods 2. NestedPCR was performed on the 2 ml of ®rst PCR mixture using 20 mM of the forward and reverse PCR primers localized at position 3372±3396 (5 0 -GGGTAATGCTAGAATGATGGCACCA-3 0 ) and 3639±3617 (5 0 -TTGGCTTTGCTGCTGTCCAGTGG-3 0 ) of human ataxin-2 cDNA and 3158± 3183 (5 0 -AGGTAATGCCAGGATGATGGCACCA-3 0 ) and 4026±4003 (5 0 . U70323).90 A. 1996). 588C (30 s) and 688C (30 s).5 mg of poly(A) 1 RNA for ®rst-strand cDNA synthesis in 20 ml reaction volume using a ThermoScript RT-PCR System kit (GIBCO BRL) at 558C for 50 min. Affaitati et al. annealing at 608C for 30 s. DNA sequence analysis Nucleotide sequencing was performed as described (Sambrook et al. according to the manufacturer's protocol. heart and liver was purchased from Origene Technologies. respectively. Total RNA extracted from peripheral blood lymphocytes of normal or SCA-2 patients was reverse transcribed using oligo-dT as primer. A splice variant of ataxin2 lacking the exon 10 has been characterized and the corresponding messenger RNA is expressed in non-neuronal tissues approximately at the same extent of full transcript.. 1998). We noticed the cloned cDNA contained a deletion of 54 bp corresponding to the complete exon 21 of ataxin-2. The ampli®cation was carried out using the following program: 2 min at 948C followed by 35 cycles at 948C (10 s). Chatsworth. Beckman automated sequencer. respectively. cDNA templates were puri®ed using QIAGEN tips columns (Qiagen.1. Ampli®ed PCR products were loaded on agarose-gel and separated by electrophoresis. 3. full length ataxin-2 transcript predominates in the brain and spinal cord. suggesting an alternative splicing of the transcript operating in vivo. ending with 5 min of extension at 688C. Human poly(A) 1-RNA from total brain. subcloned into pCR II TOPO-TA Cloning Vector (Invitrogen) and subjected to DNA sequencing. 2.3. / Gene 267 (2001) 89±93 1312 aminoacids with a predicted molecular weight of 140 kD and it is widely distributed in neuronal and non-neuronal human tissues (Pulst et al. To characterize this putative splice variant and to rule out cloning or PCR artefacts.. After an initial denaturation at 948C for 2 min. accordingly to previous studies (Sahba et al. The lower smaller band corresponds probably to the predicted splice variant. Vertical arrows indicate the positions of donor site and acceptor site. the 213 bp fragment represents a fusion between 3 0 end of exon 20 and 5 0 of exon 22. 91 and 213 bp) were visualized by etidium bromide staining. 1. in both SCA-2 (lane 2) and control cDNAs (lanes 3 and 4). Sequence analysis of the PCR fragment lacking exon 21 revealed that the alternative splice variant of ataxin-2 transcript does not affect the reading frame and connects the exon 20 and 22 with a deletion of 18 amino acids.2). the template was omitted (lane 1). we designed the isoforms with or without exon 21 as type I and type IV.A. Vertical arrows indicate the positions of donor site and acceptor site. Fig. Characterization of alternative splicing of exon 21 in human perypheral blood lymphocytes. Reverse-transcribed total RNA extracted from control (lanes 3 and 4) or SCA-2 patient (lane 2) was subjected to polymerase chain reaction using speci®c oligonucleotides primers spanning 3 0 end of exon 20 and 5 0 end of exon 22. (A) Alignment of the human ataxin-2 cDNA with its predicted aminoacid sequence of type I and type IV isoforms. Affaitati et al. corresponding to exon 21 (Fig. 2A. 1998). Thus. Exon 21 (54 bp) is absent in type IV transcript of ataxin-2. EX 20 UP and EX 22 DW indicate the oligonucleotides used for nested-PCR experiments (see Section 2. as described under Materials and Methods (see Section 2. entire exon 21 (54 bp) and 5 0 end of exon 22. The 267 bp PCR band contains the 3 0 end of exon 20. Schematic representation of alternative splice variants of ataxin-2 transcript. (C) Alignment of the aminoacid sequence of human and mouse type I ataxin-2 with aminoacid sequence lacking exon 21 (type IV). GT-AG rule that predicts the junction between donor site and acceptor site is located between exon 20 and exon 22. 2. In the control sample. To rule out artefacts of PCR reaction. In contrast. / Gene 267 (2001) 89±93 Fig.. (B) Schematic diagram of alternative splice variants (type I and type IV) of human ataxin-2.2). subcloned in pCRII TOPO-TA Cloning Vector and subjected to DNA sequence analysis. the ampli®ed products (267 and 213 bp) were puri®ed from gel. 20 ml of the PCR reactions were resolved on 2% agarose-gel and visualized by etidium bromide staining.B). respectively. The numbers indicate the nucleotide position in the ataxin-2 cDNA. A representative experiment is shown. . As shown in Fig. Shen. These data demonstrate the presence of an alternative splice variant of human ataxin-2 transcript. heart. investigated whether these variants were differentially expressed in distinct neuronal and non-neuronal human and mouse tissues. .. Mouse SCA-2 gene that lacks the exon 21 conserves reading frame downstream the splicing site as shown in Fig. McCall. Kwiatkowski Jr. 3A.. H. T. heart and placent) was subjected to polymerase chain reaction as described in Materials and Methods (see Section 2. Chun. Servadio.J. Identi®cation of alternatively spliced of exon 21 in human and mouse tissues. To this end.1). Orr. liver and were detectable at different stages (E12±E17) of mouse development (Fig. Moreover splice variant lacking exon 21 is conserved in mouse tissues. 3.A. Roth.Y. Stazione Zoologica `Anton Dohrn'. We have. cerebellum. It is not known whether these splice variants have distinct physiological functions in a particular tissue. 1998)..92 A.. A representative experiment is shown. (A) Reverse transcribed poly(A) 1 mRNA isolated from various human tissues (total brain. a clathrin-mediated trans-Golgi signal (24). 95/95'. while another clone lacks the exon 21 (MGI TC Report: TC141828).. 1996). 1994.. H. lacking the exon 21 and coding for an isoform of 2.. Avvedimento for critical reading the manuscript.. heart and liver were subjected to polymerase chain reaction as described in Materials and Methods (see Section 2. Since the exon 21 is too small to be used as probe. we were not able to perform Northern blot analysis on RNA samples.1). M. S. To date. both isoforms (type I and type IV) of ataxin-2 transcripts are expressed in human brain. Zoghbi... spinal cord. One clone corresponds to the full length sequence of mouse ataxin-2. References Ban®. Special thanks to Prof V.T. cerebellum. spinal cord. The molecular characterization of heterogeneity of isoform expression of ataxin-2 will contribute to understand the functional role. also.E. although our data cannot determine the precise quantitative ratio to isoform I. was supported by a Fellowship of the CNR Biotechnology program. on reverse transcribed RNAs extracted from several human tissues and on cDNAs from mouse tissues. (B) Total RNA from mouse embryo at E12±E17 and mouse cDNAs from brain. A.. L.. Acknowledgements We thank Raimondo Pannone (SBM. Affaitati et al. / Gene 267 (2001) 89±93 Fig. the biological signi®cance and the involvement of distinct domains of the protein in key cellular activities. 1998). 3B). 20 ml of PCR reaction were resolved on 2%-agarose gel and visualized by staining with etidium bromide. This work was supported by `Murst-CNR Biotechnology program L. heart and placent. 2C. Napoli) for technical assistance and Luigi Pianese for human tissues RNAs. Similarly both isoforms are expressed in mouse brain. Duvick. we performed polymerase chain reaction.A. the function of ataxin-2 is still poorly understood. E. Ataxin-2 is a highly basic protein with several putative functional motifs such as a caspase-3 cleavage site (Rotonda et al. Search in dbEST data base identi®ed two expressed mouse sequences that are homologous to human ataxin-2.. an endoplasmic reticulum export signal (Bannykh et al.1 kDa smaller than that previously reported protein. A.J. Y. 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