Identication of alternative splicing of spinocerebellar ataxia type 2 gene

Adelina Affaitati1, Tiziana de Cristofaro1, 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 atro-

phy (SBMA) (La Spada et al., 1991; Nakamura et al., 1994),

Huntington's disease (HD) (The Huntington's Disease

Collaborative Research Group, 1993), dentatorubropallido-

luysian 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 character-

ized by the presence of about 3540 residues of glutamine,

due to unstable expansion of CAG trinucleotide repeats in

the coding region of the gene (Housman, 1995). Polygluta-

mine expansion could cause mutant proteins to adopt altered

conformation, leading to their ubiquitination, aggregation

and cell death. Intranuclear inclusions composed of aggre-

gated proteins appear to be a common feature in the pathol-

ogy of the CAG repeat (Li and Li, 1998). We have recently

demonstrated that the length of polyglutamine, its expres-

sion, 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 domi-

nant 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). Epidemiolo-

gic 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 analy-

sis identied a major transcript of 4.5 kb and the open read-

ing frame consists of 3936 bp. Ataxin-2 is composed by

Gene 267 (2001) 8993

0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V. All rights reserved.

PII: S0378-1119(01)00402-4

www.elsevier.com/locate/gene

Abbreviations: mRNA, messenger RNA; .PCR, polymerase chain reac-

tion; poly(A)1, polyadenilated RNA; RT-PCR, reverse transcriptase-poly-

merase chain reaction

* Corresponding author. Fax: 139-081-770-1016.

E-mail address: varrone@alpha.szn.it (S. Varrone).1 Adelina Affaitati and Tiziana de Cristofaro contributed equally to the

manuscript.

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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., 1996). A splice variant of ataxin-

2 lacking the exon 10 has been characterized and the corre-

sponding messenger RNA is expressed in non-neuronal

tissues approximately at the same extent of full transcript.

In central nervous system, full length ataxin-2 transcript

predominates in the brain and spinal cord, while the splice

variant is more abundant in cerebellum (Sahba et al., 1998).

Furthermore, three isoforms of mouse homolog of SCA-2

gene have been reported: type I contains the full-length

cDNA, while type II and type III lack exon 10, or exons

10 and 11, respectively (Nechiporuk et al., 1998).

In the present work, we report a new splice variant of

human SCA-2 gene that lacks the exon 21, with conserved

reading frame downstream the splicing site. The transcript is

predicted to encode a novel isoform of ataxin-2 widely

expressed in human tissues.

2. Materials and methods

2.1. Reverse transcriptase-polymerase chain reaction (RT-

PCR)

Total RNA of control or SCA-2 patients (Sambrook et al.,

1989) was isolated from peripheral blood lymphocytes by

using the TRIZOL Reagent Kit (Life Technologies). Human

poly(A)1-RNA from total brain, spinal cord, cerebellum,

heart and placent was purchased from Clontech. Total

RNA was extracted on mouse embryonic days 1217

(E12E17) by using the TRIZOL Reagent Kit (Life Tech-

nologies). 10 ml of dT oligo were used both on 1 mg of totalRNA and 0.5 mg of poly(A)1 RNA for rst-strand cDNAsynthesis in 20 ml reaction volume using a ThermoScriptRT-PCR System kit (GIBCO BRL) at 558C for 50 min.

2.2. PCR conditions

2 ml of the rst-strand mixture were added to 50 ml of thePCR mix containing 200 mM of dNTPs and 20 mM of senseprimer and antisense primer corresponding to nucleotide

positions 14691494 (5 0-GGGAAGCAAGGGCAAAC-CAGTTAGCA-3 0) and 41654142 (5 0-TCCAGTTGGTA-GAAGCAGTAGAAG-3 0) of human ataxin-2 cDNA(GenBank Accession No. U70323), respectively.

Mouse cDNA from brain, heart and liver was purchased

from Origene Technologies. 2 ml of mouse cDNA wereadded to 50 ml of the PCR mix containing 200 mM ofdNTPs and 20 mM of sense primer and antisense primercorresponding to nucleotide positions 12421267 (5 0-GGGAAGCAAGGGCAA ACCAGTTAGCA-3 0) and39483925 (5 0-TCCGGTTGGCAGAAGCAGAGAA G -3 0) of mouse ataxin-2 cDNA (GenBank Accession No.AF041472), respectively. After an initial denaturation at

948C for 2 min, 30 cycles were repeated as follows: dena-turation at 948C for 10 s, annealing at 608C for 30 s, exten-

sion at 688C for 1 min; last cycle was extended for 5 min at688C. PCR reaction mixture was resolved on 1% agarose-gel and visualized with 10 mg/ml ethidium bromide. Nested-PCR was performed on the 2 ml of rst PCR mixture using20 mM of the forward and reverse PCR primers localized atposition 33723396 (5 0-GGGTAATGCTAGAATGATGG-CACCA-3 0) and 36393617 (5 0-TTGGCTTTGCTGC-TGTCCAGTGG-3 0) of human ataxin-2 cDNA and 31583183 (5 0-AGGTAATGCCAGGATGATGGCACCA-3 0)and 40264003 (5 0- CTGGCTTTGCTGCTGTCCGGT-GG-3 0) of mouse ataxin-2 cDNA, respectively. The ampli-cation was carried out using the following program: 2 min

at 948C followed by 35 cycles at 948C (10 s), 588C (30 s) and688C (30 s), ending with 5 min of extension at 688C. Ampli-ed products were resolved on 2% agarose-gel, puried by

using gel extraction kit (QIAGEN), subcloned into pCR II

TOPO-TA Cloning Vector (Invitrogen) and subjected to

DNA sequencing.

2.3. DNA sequence analysis

Nucleotide sequencing was performed as described

(Sambrook et al., 1989), by using CEQ 2000 DNA Analysis

System, Beckman automated sequencer, according to the

manufacturer's protocol. Sp6 and T7 oligonucleotide

primers were used. cDNA templates were puried using

QIAGEN tips columns (Qiagen, Chatsworth, CA).

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.

3. Results and discussion

To isolate full length ataxin-2 cDNA, we employed poly-

merase 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 publishedsequence (see Section 2). Sequence analysis conrmed the

identity of amplied cDNA with ataxin-2 sequence. We

noticed the cloned cDNA contained a deletion of 54 bp

corresponding to the complete exon 21 of ataxin-2, suggest-

ing an alternative splicing of the transcript operating in vivo.

To characterize this putative splice variant and to rule out

cloning or PCR artefacts, we tested the presence of this

variant transcript in the cDNA derived from several tissues.

Total RNA extracted from peripheral blood lymphocytes of

normal or SCA-2 patients was reverse transcribed using

oligo-dT as primer. Reverse transcribed cDNA was used

as template for a polymerase chain reaction with oligonu-

cleotide primers spanning 14694165 bp of SCA-2 gene.

PCR product was then subjected to second PCR by using

internal oligonucleotides primers spanning 33723639 bp of

full-length SCA-2 cDNA. Amplied PCR products were

loaded on agarose-gel and separated by electrophoresis.

As shown in Fig. 1, two fragments of different size (267

A. Affaitati et al. / Gene 267 (2001) 899390

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and 213 bp) were visualized by etidium bromide staining, in

both SCA-2 (lane 2) and control cDNAs (lanes 3 and 4). In

the control sample, the template was omitted (lane 1). The

lower smaller band corresponds probably to the predicted

splice variant. To rule out artefacts of PCR reaction, the

amplied products (267 and 213 bp) were puried from

gel, subcloned in pCRII TOPO-TA Cloning Vector and

subjected to DNA sequence analysis. The 267 bp PCR

band contains the 3 0 end of exon 20, entire exon 21 (54bp) and 5 0 end of exon 22. In contrast, the 213 bp fragmentrepresents a fusion between 3 0 end of exon 20 and 5 0 of exon22. GT-AG rule that predicts the junction between donor

site and acceptor site is located between exon 20 and exon

22, accordingly to previous studies (Sahba et al., 1998).

Sequence analysis of the PCR fragment lacking exon 21

revealed that the alternative splice variant of ataxin-2 tran-

script does not affect the reading frame and connects the

exon 20 and 22 with a deletion of 18 amino acids, corre-

sponding to exon 21 (Fig. 2A,B). Thus, we designed the

isoforms with or without exon 21 as type I and type IV,

respectively.

A. Affaitati et al. / Gene 267 (2001) 8993 91

Fig. 1. Characterization of alternative splicing of exon 21 in human peryph-

eral blood lymphocytes. Reverse-transcribed total RNA extracted from

control (lanes 3 and 4) or SCA-2 patient (lane 2) was subjected to poly-

merase chain reaction using specic oligonucleotides primers spanning 3 0

end of exon 20 and 5 0 end of exon 22, as described under Materials andMethods (see Section 2.2). 20 ml of the PCR reactions were resolved on 2%

agarose-gel and visualized by etidium bromide staining. A representative

experiment is shown.

Fig. 2. Schematic representation of alternative splice variants of ataxin-2 transcript. (A) Alignment of the human ataxin-2 cDNA with its predicted aminoacid

sequence of type I and type IV isoforms. Exon 21 (54 bp) is absent in type IV transcript of ataxin-2. Vertical arrows indicate the positions of donor site and

acceptor site. EX 20 UP and EX 22 DW indicate the oligonucleotides used for nested-PCR experiments (see Section 2.2). (B) Schematic diagram of alternative

splice variants (type I and type IV) of human ataxin-2. The numbers indicate the nucleotide position in the ataxin-2 cDNA. (C) Alignment of the aminoacid

sequence of human and mouse type I ataxin-2 with aminoacid sequence lacking exon 21 (type IV). Vertical arrows indicate the positions of donor site and

acceptor site.

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Search in dbEST data base identied two expressed

mouse sequences that are homologous to human ataxin-2.

One clone corresponds to the full length sequence of mouse

ataxin-2, while another clone lacks the exon 21 (MGI TC

Report: TC141828). Mouse SCA-2 gene that lacks the exon

21 conserves reading frame downstream the splicing site as

shown in Fig. 2C.

We have, also, investigated whether these variants were

differentially expressed in distinct neuronal and non-neuro-

nal human and mouse tissues. To this end, we performed

polymerase chain reaction, as described above, on reverse

transcribed RNAs extracted from several human tissues

and on cDNAs from mouse tissues. As shown in Fig.

3A, both isoforms (type I and type IV) of ataxin-2 tran-

scripts are expressed in human brain, spinal cord, cerebel-

lum, heart and placent. Similarly both isoforms are

expressed in mouse brain, heart, liver and were detectable

at different stages (E12E17) of mouse development (Fig.

3B). Since the exon 21 is too small to be used as probe, we

were not able to perform Northern blot analysis on RNA

samples.

These data demonstrate the presence of an alternative

splice variant of human ataxin-2 transcript, lacking the

exon 21 and coding for an isoform of 2.1 kDa smaller

than that previously reported protein. This splice variant is

expressed in several human tissues, although our data

cannot determine the precise quantitative ratio to isoform

I. Moreover splice variant lacking exon 21 is conserved in

mouse tissues. Another splice variant of human ataxin-2 that

lacks the exon 10 has been recently found (Sahba et al.,

1998). It is not known whether these splice variants have

distinct physiological functions in a particular tissue.

To date, the function of ataxin-2 is still poorly under-

stood. Ataxin-2 is a highly basic protein with several puta-

tive functional motifs such as a caspase-3 cleavage site

(Rotonda et al., 1996), a clathrin-mediated trans-Golgi

signal (24), an endoplasmic reticulum export signal

(Bannykh et al., 1998) and RNA splicing motifs (Sm1 and

Sm2) (Neuwald and Koonin, 1998). The molecular charac-

terization of heterogeneity of isoform expression of ataxin-2

will contribute to understand the functional role, the biolo-

gical signicance and the involvement of distinct domains

of the protein in key cellular activities.

Acknowledgements

We thank Raimondo Pannone (SBM, Stazione Zoologica

`Anton Dohrn', Napoli) for technical assistance and Luigi

Pianese for human tissues RNAs. Special thanks to Prof

V.E. Avvedimento for critical reading the manuscript.

A.A. was supported by a Fellowship of the CNR Biotech-

nology program. This work was supported by `Murst-CNR

Biotechnology program L. 95/95'.

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A. Affaitati et al. / Gene 267 (2001) 899392

Fig. 3. Identication of alternatively spliced of exon 21 in human and mouse tissues. (A) Reverse transcribed poly(A)1 mRNA isolated from various human

tissues (total brain, spinal cord, cerebellum, heart and placent) was subjected to polymerase chain reaction as described in Materials and Methods (see Section

2.1). 20 ml of PCR reaction were resolved on 2%-agarose gel and visualized by staining with etidium bromide. A representative experiment is shown. (B) Total

RNA from mouse embryo at E12E17 and mouse cDNAs from brain, heart and liver were subjected to polymerase chain reaction as described in Materials and

Methods (see Section 2.1). 20 ml of PCR reaction were resolved on 2%-agarose gel and visualized by staining with etidium bromide.

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