Identification and expression of a mouse ortholog of A2BP1
* Hiroki Shibata,
** Tramy Vo,
Duong P. Huynh,
Rose Moss Laboratory for Parkinson and Neurodegenerative Diseases, Burns and Allen Research Institute, Cedars-Sinai Medical Center,
Los Angeles, California 90048, USA
Division of Neurology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California, USA
Received: 30 January 2001 / Accepted: 6 April 2001
Abstract. Human ataxin-2 contains a polyglutamine repeat that is
expanded in patients with spinocerebellar ataxia type 2 (SCA2).
Ataxin-2 is highly conserved in evolution with orthologs in mouse,
Caenorhabditis elegans, and Drosophila melanogaster. It interacts
at its C-terminus with ataxin-2 binding protein 1, A2BP1. This
study presents a highly conserved mouse ortholog of A2BP1, des-
ignated A2bp1. The amino acid sequence of the human and mouse
protein is 97.6% identical. This remarkable degree of conservation
supports the fact that these proteins have an important basic func-
tion in development and differentiation. Sequence analysis reveals
the existence of RNA binding motifs. The A2bp1 transcript was
found in various regions of the CNS including cerebellum, cerebral
cortex, brain stem, and thalamus/hypothalamus. The A2bp1 pro-
tein was detected by immunocytochemistry in the CNS and con-
nective tissue of the mouse embryo starting at stage E11, as well
as in the heart at all stages. Mouse embryos showed varying ex-
pression of A2bp1 at all stages. Previous studies in other model
systems had implicated the orthologs of ataxin-2 and A2BP1 in
development. This study suggests a role for A2bp1 in embryogen-
esis as well as in the adult nervous system, possibly mediated by
a function in RNA distribution or processing.
Unstable triplet repeats have been identified as the cause of several
hereditary neurodegenerative diseases. A CAG repeat expansion
encoding a polyglutamine stretch has been found in eight dominant
neurodegenerative diseases such as Huntington disease (The Hun-
tington’s Disease Collaborative Research Group 1993), spinobul-
bar muscular atrophy (SBMA; La Spada et al. 1991) and the spi-
nocerebellar ataxias (SCA1: Orr et al. 1993; SCA2; Pulst et al.
1996; SCA3: Kawaguchi et al. 1994; SCA6: Zhuchenko et al.
1997; SCA7: David et al. 1997).
In contrast to the widespread expression of the proteins impli-
cated in polyglutamine disease, neuropathology is typically re-
stricted to a few cell types. This phenomenon has led to the search
for interacting partners. We have recently isolated a protein that
interacts with ataxin-2, the gene product of the human spinocer-
ebellar ataxia type 2 (SCA2) gene (Shibata et al. 2000). This
protein, designated A2BP1 for a
taxin-2 binding protein 1,isa
member of a novel family of putative RNA-binding proteins.
A2BP1 and ataxin-2 are highly conserved among species. Mouse
A2BP1 has 30.1% amino acid identity and 51.1% similarity with
its Caenorhabiditis elegans ortholog, the sex-determination gene
The expression of the A2BP1 transcript is indeed restricted to
particular brain regions including the cerebellum. It was also de-
tected in heart and muscle tissues. In contrast to most of the other
proteins implicated in polyglutamine diseases, ataxin-2 and
A2BP1 are cytoplasmic rather than nuclear proteins (Huynh et al.
2000). They colocalize with markers of the trans-golgi network
and show a juxtanuclear immunofluorescence pattern (Shibata et
We have previously used RNA interference in C. elegans to
examine the function of worm orthologs of ataxin-2 and A2BP1,
named atx-2 and fox-1 respectively (Kiehl et al. 2000). These
genes play an essential role in early embryonic development. They
are expressed in the germline and embryo of the worm. Fox-1 is a
known sex-determinant gene (Hodgkin et al. 1994). It also func-
tions as a numerator element that reduces the effect of the extra X
Chromosome (Chr) in the hermaphrodite to a haploid dose effect.
No human deletions or loss of function mutations involving either
gene are known.
A putative ancestral gene must have existed before the diver-
gence of humans and nematodes from a common ancestor. It likely
duplicated and diverged into the mammalian A2BP1 and RBM9 as
well as the worm genes fox-1, R74.5, and ZC404.8 and the dro-
sophila gene CG18441 (Fig. 1A,B).
This study has identified and characterized the murine ortholog
of A2BP1, termed A2bp1. Its protein sequence is highly similar to
the human protein. Different transcripts and protein sizes were
found in the mouse, similar to human isoforms. We have charac-
terized the regional expression pattern for certain CNS regions as
well as for other organs. A characteristic embryonic expression
pattern was found for A2bp1. The unusually high overall conser-
vation of A2bp1 suggests that it is evolutionarily stabilized for a
specific cellular function, possibly mediated by its RNA binding
Materials and methods
A mouse ortholog of A2BP1 was identified by screen-
ing a lambda ZAP II newborn mouse brain library (Stratagene, La Jolla,
Calif.) by using the human A2BP1 cDNA insert fragment (Shibata et al.
2000) as a probe. The procedures of library screening followed the sup-
plier’s protocol (Stratagene).
Northern blot analysis.
Total RNA samples were extracted from dif-
ferent regions of mouse brain and rat cerebellum with Trizol reagent
(GibcoBRL, Rockville, Md.). The RNA was electrophoresed through 1.2%
agarose gel and blotted onto GeneScreen Plus membrane (NEN Life Sci-
ence, Boston, Mass.). An EcoRI 0.7-kb fragment of human A2BP1 was
used as a probe. It was labeled with ␣-[
P]dCTP by RadPrime random
priming kit (GibcoBRL). The conditions of hybridization and washing
followed the supplier’s protocols (NEN). The relative loading and integrity
* Both authors contributed equally
Correspondence to: S.M. Pulst; E-mail: email@example.com
** Present address: Division of Disease Genes, Institute of Genetic Infor-
mation, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-
Mammalian Genome 12, 595–601 (2001).
© Springer-Verlag New York Inc. 2001