Gene 267 (2001) 8993

www.elsevier.com/locate/gene

Identication 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 3540 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: varrone@alpha.szn.it (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 identied 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

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

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 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.
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 (RTPCR)
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 Technologies). 10 ml of dT oligo were used both on 1 mg of total
RNA and 0.5 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.
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 14691494 (5 0 -GGGAAGCAAGGGCAAACCAGTTAGCA-3 0 ) and 41654142 (5 0 -TCCAGTTGGTAGAAGCAGTAGAAG-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 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 12421267 (5 0 GGGAAGCAAGGGCAA ACCAGTTAGCA-3 0 ) and
39483925 (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: denaturation 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 at
688C. PCR reaction mixture was resolved on 1% agarosegel and visualized with 10 mg/ml ethidium bromide. NestedPCR was performed on the 2 ml of rst PCR mixture using
20 mM of the forward and reverse PCR primers localized at
position 33723396 (5 0 -GGGTAATGCTAGAATGATGGCACCA-3 0 ) and 36393617 (5 0 -TTGGCTTTGCTGCTGTCCAGTGG-3 0 ) of human ataxin-2 cDNA and 3158
3183
(5 0 -AGGTAATGCCAGGATGATGGCACCA-3 0 )
and 40264003 (5 0 - CTGGCTTTGCTGCTGTCCGGTGG-3 0 ) of mouse ataxin-2 cDNA, respectively. The amplication was carried out using the following program: 2 min
at 948C followed by 35 cycles at 948C (10 s), 588C (30 s) and
688C (30 s), ending with 5 min of extension at 688C. Amplied 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 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). 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, suggesting 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 oligonucleotide 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) 8993

Fig. 1. 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 specic oligonucleotides primers spanning 3 0
end of exon 20 and 5 0 end of exon 22, as described under Materials and
Methods (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.

91

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 (54
bp) and 5 0 end of exon 22. In contrast, the 213 bp fragment
represents a fusion between 3 0 end of exon 20 and 5 0 of exon
22. 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 transcript does not affect the reading frame and connects the
exon 20 and 22 with a deletion of 18 amino acids, corresponding to exon 21 (Fig. 2A,B). Thus, we designed the
isoforms with or without exon 21 as type I and type IV,
respectively.

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

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.

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-neuronal 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 transcripts are expressed in human brain, spinal cord, cerebellum, 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 understood. Ataxin-2 is a highly basic protein with several putative 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 characterization of heterogeneity of isoform expression of ataxin-2
will contribute to understand the functional role, the biological 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 Biotechnology program. This work was supported by `Murst-CNR
Biotechnology program L. 95/95'.

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