From the Laboratoire de Génétique et
Physiologie du Développement, CNRS, INSERM, Université
de la Méditerranée, Luminy Case 907, 13288 Marseille Cedex
9, France, § Centre de Recherches en Biochimie
Macromoléculaire, CNRS, 34293 Montpellier Cedex 5, France,
Centre d'Immunologie de Marseille-Luminy, CNRS, INSERM,
Université de la Méditerranée, 13288 Marseille Cedex
9, France, and ** National Institute of Genetics, Mishima,
Shizuoka 411-8540, Japan
Received for publication, October 17, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Eukaryotes possess multiple isoforms of the
a subunit of the V0 complex of vacuolar-type
H+-ATPases (V-ATPases). Mutations in the V-ATPase
a3 isoform have recently been shown to result in
osteopetrosis, a fatal disease in humans, but no function has yet been
ascribed to other isoforms. In Caenorhabditis elegans, the
unc-32 mutant was originally isolated on the basis of its
movement defect. We have isolated four new mutant alleles, the
strongest of which is embryonic lethal. We show here that
unc-32 corresponds to one of the four genes encoding a
V-ATPase a subunit in the nematode, and we present their
expression patterns and a molecular analysis of the gene family.
unc-32 gives rise via alternative splicing to at least six
transcripts. In the uncoordinated alleles, the transcript
unc-32 B is affected, suggesting that it encodes an isoform
that is targeted to synaptic vesicles of cholinergic neurons, where it
would control neurotransmitter uptake or release. Other isoforms
expressed widely during embryogenesis are mutated in the lethal alleles
and would be involved in other acidic organelles. Our results indicate
that V-ATPase a subunit genes are highly regulated and have
tissue-specific function.
Vacuolar-type H+-ATPases
(V-ATPases1) are
ATP-dependent proton pumps involved in the acidification of
intracellular compartments in eukaryotic cells and are important for
many facets of cellular and organismal function (for review, see Refs.
1-3). As well as contributing to fundamental cellular processes in the
majority of eukaryotic cells, they also play specialized functions in
certain cell types. In macrophages, the progressive acidification of
phagosomes is an integral part of their maturation into lysosomes,
whereas the acidification of early endocytic compartments is necessary for the recycling to the plasma membrane of transport molecules such as
the transferrin receptor. In neurons, neurotransmitter trafficking is
dependent on V-ATPases. The H+-driven antiporters
responsible for synaptic vesicle import of a specific neurotransmitter,
such as the vesicular acetycholine transporter, are known to be driven
by proton gradients established by a V-ATPase localized to the same
vesicle (4, 5).
The V-ATPases are composed of two complexes, V1 and
V0, that serve different functions (for review, see Refs.
6, 7). V1, a complex of 570 kDa composed of eight subunits
(A-H), is located on the cytoplasmic side of the vesicle
membrane and is responsible for ATP hydrolysis. V0 is a
260-kDa complex with five different subunits (a,
d, c, c', and c") of
17-100 kDa. It is an integral membrane complex and is responsible for
proton translocation from the cytoplasm to the lumen of the vesicle. In
essence, the V-ATPases resemble, both in terms of sequence and
structure, the ATP synthases of mitochondria. ATP synthases, however,
couple the synthesis of ATP to the flow of protons from the
mitochondrial matrix to the cytoplasm, and the overall topology of its
two components is inverted, with the F1 complex (analogous
to the V1 complex) being on the lumenal side.
Interestingly, there is no clear homologue for the V0
100-kDa a subunit within the analogous F0
complex, suggesting that it plays a specific role in the function of
the V-ATPases. In yeast, there are two a subunit genes that
encode proteins that have distinct subcellular localization (8). One, VPH1p, is associated with the central vacuole, whereas STV1p is thought
to be a Golgi/endosome-specific form. Thus the two a
subunits may be involved in targeting yeast V-ATPases to different
intracellular membranes. Three murine a subunit genes have
been characterized (a1, a2, and a3;
Ref. 9). For a1 and a3, there are two and three
alternative transcripts, respectively, giving the possibility of
substantial variation in the structure of the murine V-ATPase. Intriguingly, the level of expression of the different transcripts of
the murine genes varies among different tissues (9). This suggests that
the V-ATPase complexes may have specialized compositions and
structures, not only at the intracellular level but also in different
tissues. The recent characterization of knockout mice for the
a3 gene (ATP6i), which exhibit severe osteopetrosis due to
loss of osteoclast-mediated extracellular acidification but no general
defect in V-ATPase function, supports such a hypothesis (10). More
recently, mutations in the human orthologue have been shown to be
responsible for a subset of human osteopetrosis (11, 12).
Here, we describe the genetic and molecular identification of the
Caenorhabditis elegans unc-32 gene, which encodes a set of
V-ATPase 100-kDa a subunits. We show that the gene is one of a family of four V-ATPase a subunits encoding genes
expressed in C. elegans. Furthermore, we show that a number
of alternative unc-32 transcripts are produced by
differential splicing of alternative exons and have characterized five
mutants, two of which affect specifically one isoform. On the basis of
our phenotypic and molecular analysis of the mutants, we postulate that
specific isoforms may be involved in nervous system function, whereas
others are required for viability.
Strains and Mutagenesis--
All strains including the wild-type
N2 Bristol and strains containing the unc-32 alleles and the
transformants were grown and maintained at 20 °C as described (13).
Four new ethyl methanesulfonate-induced alleles,
unc-32(f120), unc-32(f121), unc-32(f123), and
unc-32(f131), were isolated in two noncomplementation
screens for new unc-32 alleles. The three former were
induced on a dpy-17(e164) ncl-1(e1865) chromosome, the
latter on an unc-36(e251) chromosome. Several thousand
mutagenized genomes were tested against unc-32(e189) and
scored for an uncoordinated phenotype. The new alleles were back-crossed at least four times to wild type, and genetic analysis was
conducted using standard procedures. The two alleles, f121 and f123, were homozygous lethal and were kept heterozygous
with the balancer qC1, dpy-19 glp-1, provided by J. Kimble.
The integrated transformant eIs16[ZK637,
unc-32+] was kindly provided by J. Sulston. The nonsense
suppressor sup-7(st5) (14) as well as the
smg-2(e2008) suppressor involved in mRNA surveillance
(15, 16) was combined with all alleles but f131.
Plasmid Constructions--
All the plasmids used in this work
were derived from the A1E5 plasmid (a gift from M. Craxton). A1E5 is a
partial SauIIIA restriction fragment of the ZK637 cosmid
(GenBank accession number Z11115), from 19434 to 32570, cloned in
BlueScribe. E5-NS was obtained after NheI and
SpeI restriction and religation. E5-BK was constructed by a
partial BamHI restriction to keep the BamHI site
of the large exon of ZK637.8. E5 Transgenic Lines--
Plasmids for injection were prepared by a
standard alkaline lysis procedure, followed by LiCl precipitation.
Injections were performed as described (17). Subclones of ZK637 were
injected at a concentration of 10 ng/µl with plasmid pRF4 (encoding
rol6(su1006), which confers a dominant rolling phenotype) at
a concentration of 100 ng/µl. Some of the transformants also received
either plasmid pPD93.97 (encoding a GFP reporter gene driven by the
myo-3 promoter) or pPD20.97 (encoding a LacZ reporter gene
driven by the myo-2 promoter) at a concentration of 20 ng/µl to check the presence of coinjected DNA directly by screening
GFP in body wall muscle or Transcript Analysis--
Partial sequences from 15 cDNAs
were aligned on the genome, and three clones were fully sequenced
(yk15a11, yk244c11, and yk5d7). There was evidence for alternative
splicing, and the alternative transcripts from the three clones were
designated as unc-32 A, B, and C, respectively. The
differential splicing patterns were also analyzed by restriction
digestion and/or sequencing of specific reverse
transcription-polymerase chain reaction (RT-PCR) products. Total RNA
from mixed populations of wild-type worms was prepared as previously
described (18), reverse transcribed, and subjected to PCR with various
sets of primers as well as the transpliced leaders SL1 and SL2 (19,
20). Amplification conditions and primers are available on request. The
RT-PCR products were analyzed by restriction using enzymes cutting the
transcripts only once, in exon 3 (XhoI), exon 4a
(EcoRI), exon 4b (PstI), exon 7a
(PvuI), and exon 7b (DrdI). Some RT-PCR products
were sequenced on an ABI machine using dye deoxyterminators (Prism;
PerkinElmer Life Sciences). The traces were analyzed using the
acembly program, developed by J. Thierry-Mieg and M. Potdevin.
When referring to the ZK637 cosmid, exon positions are: exon 1, 21653-21816 (coding sequence starting at 21664); exon 2, 21898-22074; exon 3, 22182-22310; alternative exon 4a, 22560-22714; alternative exon 4b, 23003-23109; alternative exon 4c, 23347-23468; exon 5, 23629-23838; exon 6, 23893-25147; alternative exon 7a, 25269-25391; alternative exon 7b, 26117-26257; exon 8, 26370-26589; exon 9, 26754-26925; and exon 10, 27145-27250. Transcripts were transpliced with SL1 at position 21653. Alternative polyadenylation occurs at
similar frequencies at positions 27671 (atypical poly(A) signal agtaaa
16-11 bp before the poly(A)) or 27784 (atypical poly(A) signal aacaaa
17-12 bp before the poly(A)). Note that the existence, extent, and
alternative status of exons 4a, 4b, 4c, 7a, and 7b were wrongly
predicted for ZK637.8 in the data bases.
In Situ and GFP Expression Analyses--
In situ hybridization
analyses were performed as previously described (21). Transgenic worms
carrying the unc-32e4::GFP construct were observed
with a confocal microscope; the image in Fig. 6G is the
projection of several Z serial sections.
Mutant Analysis--
The genomic DNA of four mutant alleles (all
but f123) was amplified with Takara Taq. PCR
products covering the segments from Sequence Comparisons--
The nematode VHA-5 and VHA-6 proteins
correspond respectively to the predicted F35H10.4 (GenBank accession
number AAA81682) and VW02B12.1 (GenBank accession number CAA90758)
proteins, encoded by the expressed YK6569 and YK634 genes. The nematode VHA-7 protein, corresponding to the predicted C26H9A.1 protein (GenBank
accession number T19492), encoded by the YK778 expressed gene, was
edited by removing the first 291 amino acids and the next to last exon,
which does not occur in 5 of 5 independent cDNA clones. The 5'
region removed does not correspond to any available cDNA, nor does
it resemble sequence in any of the well characterized 100 kDa
a subunit proteins. Comparisons between the available human
genomic sequence (GenBank accession number AC002537) and the published
cDNA sequence for the human a1 gene (GenBank accession
number NM_005177) allowed the deduction of the intron-exon boundaries.
The intron-exon boundaries for the human a3 gene (OC116)
were published previously (22). Amino acid sequences were aligned with
the Multiple Alignment Program (developed by X. Huang).
Phylogenetic trees were produced using the full-length protein
sequences by neighbor joining using Clustalw alignment in Phylip format
and the Treeview program.
Three unc-32 Alleles Are Viable but Uncoordinated--
The
unc-32(e189) allele was isolated by Brenner (13) as part of
the first genetic screen for C. elegans mutants. Homozygous mutants are healthy and of normal size but exhibit a ventral coiler phenotype during backward movement (Fig.
1). This defect is seen from the L2 stage
onward. The only additional defect observed in e189 is the
brood size, smaller by one-third when compared with wild type, probably
because of a reduction in number of fertile sperm. We carried out a
genetic noncomplementation screen and isolated two more recessive
viable unc-32 alleles, f120 and f131. Although f120 shows a similar phenotype to e189
and was subsequently found to correspond to an identical molecular
lesion (see below), f131 presents a much stronger
uncoordinated (Unc) phenotype. The L1 larvae were paralyzed and kinky
as soon as they hatched; adults moved slightly better but were extreme
forward and backward coilers (Table I).
In addition, f131 animals were small, thin, transparent, and
slow-growing and had a brood size half that of the wild type. From the
interactions with the nDF17 deletion, fully uncovering the
region, that lead to a stronger phenotype, i.e. a high
frequency of lethals (Table I), and the molecular evidence presented
below, we believe that these three uncoordinated alleles, e189,
f120, and f131, are partial loss-of-function
alleles.
Two unc-32 Alleles Are Lethal, and the Locus Is Complex--
The
two further alleles, f121 and f123, isolated in
the screen are zygotic lethal. f123 is equivalent to the
deficiency in all interallelic combinations and therefore behaves as a
null (Table I). Additionally, it is suppressible by the amber nonsense suppressor sup-7 (14), suggesting the presence of a
premature stop codon. f123 homozygotes arrest as late
embryos, often with vacuolization of the intestinal region. This null
allele is associated with a strict maternal effect lethality, giving
rise to severe cell adhesion defects in the early embryos. In addition,
we cannot exclude a sterility owing to lack of production of mature
gametes. The weaker lethal allele f121 arrests at the larval
L2, L3, or L4 stage, and before their premature death the movement of
the larvae appears close to that of wild type. In contrast to
f123, f121 almost fully complements the mild Unc
alleles e189 and f120 and is fully viable when
combined with e189, f120, and f131. f121/f131 larvae move better than f131 homozygotes but are still
highly impaired in their locomotion. Adults, however, are only backward ventral coilers, just like e189. Hence, the lethality of
f121, unlike that of f123, is rescued by each of
the three Unc alleles, and conversely the Unc phenotype of these three
alleles is partly rescued by f121. Such interallelic
complementation defines unc-32 as a complex locus, a
property often associated with alternative splicing.
unc-32 Encodes a 100-kDa a Subunit of V-ATPase--
Genetic
mapping had previously placed unc-32 between
sma-2 and lin-12 in the center of LG
III (13). Transformation rescue of unc-32(e189) by
microinjection of cosmids from the candidate region of the physical map
showed that the gene is contained within both ZK637 and
T05B22 (Fig.
2A). We further refined the
position of the gene by transformation rescue to a region of 9 kilobases (E5-BK, E5-NS, and E5-lin-9 constructs; see Fig.
2B), corresponding to the predicted gene ZK637.8. This gene
encodes a protein similar in sequence to V-ATPase 100 kDa a
subunits. Constructions in which part of ZK637.8 was deleted or in
which a premature stop codon was introduced into the coding sequence of
the gene failed to rescue the uncoordinated phenotype (E5-
Sequencing of the ZK637.8 genomic region from four mutant alleles
revealed different molecular alterations: e189 and
f120 contain a point mutation in the splice acceptor site of
exon 4b, f131 in the splice donor site of exon 6 (see
below), and f121 is a G to A transition in exon 6 (Table I).
Together, these results confirm the identity of unc-32 as
ZK637.8. The gene is the first in an operon containing two others,
ZK637.9 and ZK637.10. The former encodes a protein similar in sequence
to thiamine pyrophosphokinase (24). The latter encodes a putative
mitochondrial thioredoxin reductase. By RT-PCR, we showed that these
three genes are expressed, and that although the transcript of ZK637.8
is trans-spliced to SL1, both ZK637.9 and ZK637.10 can be trans-spliced
to SL2 (data not shown; Ref. 25), as is normally the case for genes in
C. elegans operons (26). Although in many operons the genes
are functionally related, no evident link is apparent so far between the three genes of the unc-32 operon.
unc-32 Transcripts Are Alternatively Spliced--
Sequencing of
existing cDNAs and specific RT-PCR products revealed a number of
alternative splice patterns for the unc-32 transcripts (Fig.
3, A and B). All
unc-32 transcripts analyzed contain 10 exons. There are at
least six different transcripts, using three variants for exon 4 (a-c)
and two for exon 7 (a and b). Although the alternative exons 4a-c do
not encode peptides that closely resemble sequences found in other
V-ATPases, they occur at a position in the sequence where an
alternative splicing pattern has also been observed for the murine
a1 gene (9), suggesting that
they may have an important functional role. Significantly, the mutation
in unc-32(e189) is a G to A transition in the consensus splice acceptor site of exon 4b, meaning that in this allele, only the
alternative transcripts with exon 4b should be affected. Consistent
with this, anomalous unc-32 transcripts were found by RT-PCR
amplification of the fourth exon region of total RNA extracted from
unc-32(e189) homozygotes (Fig. 3C). Cloning and sequencing of the forms that contain exon4b in e189
homozygotes revealed the addition of 22 bp upstream of the exon 4b
because of the use of a new acceptor splicing site. The resulting
anomalous transcript contains a premature stop codon and would encode a nonfunctional protein. The other alternative forms containing exons 4a
and 4c are still produced (Fig. 3C).
The alternative seventh exons, 7a and 7b, are similar in sequence and
presumably arose from a recent duplication, because a single exon of
similar sequence is present in other species (Fig. 3A). The
f131 allele contains a G to A transition at the consensus
splice donor site of the sixth exon, which affects the splicing of
these alternative seventh exons (Fig. 3C). The large anomalous unc-32 transcript found by RT-PCR amplification of
the seventh exon region of total RNA extracted from
unc-32(f131) homozygotes was sequenced. This revealed the
presence of intron 6 between exon 6 and exon 7a. Because a stop codon
is present in the sixth intron, the resulting mutant protein would be
truncated in the eighth transmembrane domain. The other band with a
size similar to that of wild type (Fig. 3C) corresponds to a
mixture of alternate transcripts using cryptic donor sites. Because
this allele is a hypomorph, not a null, we expect that some of these
transcripts can be translated into a near full-length product that
would differ from the wild type only in the relatively poorly conserved
region around the exon 6-exon 7 junction.
V-ATPase a Subunits Are a Large and Highly Conserved
Family--
UNC-32 belongs to a family of highly conserved V-ATPase
a subunit proteins. In other species there are multiple
genes encoding V-ATPases a subunits. There are three genes
in mice (9) and other vertebrates, and searches in the available
Drosophila genomic sequence indicate that there are at least
four members in the fly.3 In C. elegans, in addition to
UNC-32, there are three other members of the family, named VHA-5,
VHA-6, and VHA-7. Each of the four genes has a distinctive intron-exon
structure. We could not detect alternative splicing of these genes, as
judged by the analysis of several
cDNAs.4 The existence of shared intron-exon
boundaries among the four nematode genes and the two human genes for
which data are available supports their common ancestry (Fig.
4A). Each of these putative proteins contain nine potential
membrane-spanning regions (Ref. 27 and Fig. 4B). The
sequence identity (and similarity) between UNC-32A and other nematode
and murine a subunit proteins are as follows: VHA-5, 50%
(67%); VHA-6, 50% (65%); VHA-7, 46% (62%); a1, 55% (69%); a2,
43% (60%); and a3, 37% (55%). Phylogenetic analysis of the
different members of this family indicates, in agreement with previous
suggestions based on a more limited analysis (9), that the multiple
genes appeared independently in vertebrates and invertebrates
subsequent to their divergence from a common ancestor (Fig.
5). Interestingly, although the UNC-32
protein is closer in sequence to the vertebrate a1 protein
than to the other nematode proteins, the vha-7 gene shares
more intron-exon boundaries with the human a1 and
a3 genes than other nematode genes.
V-ATPase a Subunits Have Specific Expression Domains--
The
expression pattern of each of the four V-ATPase a genes in
C. elegans was analyzed by in situ hybridization.
Two of the genes, vha-6 and vha-7, have very
limited and nonoverlapping domains of expression, whereas the two
others, unc-32 and vha-5, are expressed more
broadly, yet each with a distinctive pattern (Fig.
6). Transcripts for unc-32
were detected at high levels in early embryos (Fig. 6A) from
the one-cell stage to the end of gastrulation. In larvae and adults,
transcripts were clearly present in the gonad (Fig. 6, C and
D), in the intestine, in many neurons in the head, and in
motoneurons in the ventral cord (Fig. 6D). This expression pattern is consistent with the expression of a LacZ reporter gene driven by the upstream region of the
unc-32 gene (28).5 Similarly,
vha-5 showed precocious and widespread embryonic expression (Fig. 6E), followed by a more restricted pattern in larvae
and adults. mRNAs are present in many tissues where
unc-32 is also expressed, but the detailed patterns were
different, because the gene is also expressed in the excretory cell and
canals (Fig. 6F) and probably some somatic gonad cells (Fig.
6, G and H). In contrast, expression of
vha-6 started late, in the intestine of the 2-fold embryo
(Fig. 6I). It was strong but limited to the intestinal cells
at all stages (Fig. 6, J-L). The vha-7 gene was silent during embryogenesis and the first larval stages (Fig. 6M). Its expression was first observed at the L3 stage and
was limited to areas of the developing and mature gonad corresponding to the spermatheca (Fig. 6N) and the region where meiotic
oocytes are in the pachytene and early diakinesis stages. It vanished abruptly in mature oocytes (Fig. 6, O and P).
Although the patterns of expression of these other genes are
suggestive, their functions remain to be investigated.
unc-32 Transcripts Containing Exon 4b Are Specifically Expressed in
the Nervous System--
In situ hybridization with the
unc-32 gene revealed a large domain of expression,
especially in the embryo where the expression is ubiquitous. To test
the hypothesis that unc-32 alternative transcripts have
tissue-specific expression patterns, we made a fusion construct,
unc-32e4::GFP, in which GFP was inserted into the
alternative exon 4b. GFP expression in wild-type strains carrying this
construct was restricted to the nervous system (Fig. 6Q), including all the motoneurons in the ventral cord, the dorsal, the
lateral, and all the sublateral cords, PLML/R, PVM, ALML/R, AVM,
ALNL/R, PLNL/R, PVDL/R, PDEL/R, SDQL/R, and almost all the neurons in the head.
unc-32(e189) Is Affected in the Motor Circuit--
The analysis of
the uncoordinated phenotype of unc-32(e189) suggested a
possible defect in specific classes of motoneurons (29). Serial
reconstruction of the anterior part of the ventral nerve cord behind
the retrovesicular ganglion in the mutant, however, failed to show any
wiring defect in the motoneurons. They revealed instead that synaptic
vesicles had an abnormal and distinctive morphology
compared with wild
type.6 This strongly suggests that the function
rather than the structure of the motor circuit is compromised in
unc-32(e189) mutants. Consistent with this, mutant animals
are resistant, at a low but consistent level, to inhibitors of
acetylcholine esterase, as are mutants in other genes involved in
acetylcholine synthesis, packaging, release, or signal reception (30).
We have unambiguously identified unc-32 as being a nematode
V-ATPase a subunit and show that unc-32 is
expressed in some motoneurons and is thus likely to be directly responsible for the uncoordinated phenotype. We propose that in the
unc-32(e189) mutant, V-ATPases are not functional in
motoneurons, leading to a lack of import of the acetylcholine
neurotransmitter into synaptic vesicles, a function that as been
previously suggested for V-ATPase (4, 5).
Alternative Splice Forms Are Expressed in Motoneurons--
There
are at least six alternative transcripts for the unc-32
gene. The unc-32(e189) mutation affects the generation of
two of the six transcripts, namely those containing exon 4b. In other terms, in the e189 allele, there is no transcript with exon
4b, or if a transcript contains exon 4b, it is nonfunctional. One hypothesis would be that the isoforms with exon 4b are required for
synaptic vesicle function in motoneurons but are dispensable in other
tissues. Alternatively, motoneurons could force the splicing apparatus
to always include exon 4b, thus generating a null allele in this cell
type. The fact that a GFP fusion in exon 4b is specifically expressed
in the nervous system favors the latter hypothesis.
Other Alternative Splice Forms Are Required during
Development--
The unc-32(f121) and the null
unc-32(f123) alleles are lethals, so certain UNC-32 isoforms
must be absolutely required for viability. Because
unc-32(f121) corresponds to a point mutation in an exon
common to all isoforms and will affect all alternative unc-32 transcripts, it remains to be determined which UNC-32
isoforms have an essential function. The lethality of those alleles is, however, consistent with the fact that unc-32 is widely
expressed in early embryogenesis. Rescue of the e189
uncoordinated phenotype by the f121 lethal allele suggests
that at least one of the f121 mutant forms is partially
functional in the synaptic vesicles of the motor circuit. The
f121 mutation results in a Glu for Gly substitution in the
third transmembrane domain at a position that is not fully conserved
between species. In motoneurons, this substitution in transcripts
containing exon 4b might not perturb the function of the V-ATPase.
Our results have revealed that the function and regulation of
unc-32 are complex. First, there are at least six
alternative transcripts for this gene. Second, the three uncoordinated
alleles are affected in the generation of these alternative
transcripts. Third, the different mutant alleles display distinct
phenotypes and unexpected interallelic complementation. These could be
due to perturbations of an equilibrium between alternatively spliced forms of unc-32, each of which would encode a protein with a
specific function. The smg-2 suppressor is involved in
mRNA surveillance (15) and has been shown to modulate the level of
alternatively spliced mRNAs (16). Significantly, the lethality of
f121 was rescued by smg-2, but
unc-32(f121); smg-2(e2008) adults did not lay eggs (data not shown).
Four V-ATPase a Subunits in the Nematode--
The
unc-32 gene corresponds to one of four nematode V-ATPase
a subunits. Just like in vertebrates, where different
a subunit isoforms are targeted to different tissues (9,
31), each of the C. elegans V-ATPase a genes has
a specific, largely nonoverlapping, domain of expression.
Tissue-specific expression of alternatively spliced forms of the murine
a1 gene has also been reported (9). Furthermore, in yeast,
the two a subunit isoforms have been implicated in the
differential intracellular localization of the V-ATPase complex (8).
This suggests that different isoforms of the a subunit,
arising from alternative splicing or the expression of different genes,
or both, have specific patterns of expression, both in terms of tissue
distribution and targeting to individual intracellular membrane
compartments. In the nematode, further variation of the structure of
the V-ATPase is possible, because three c subunit genes
(vha-1, -2, and -3) have been
described (32-34). Given their conservation through evolution, future
studies using C. elegans may allow a deeper understanding of
the enormous potential for variation in the structure, function, and
regulation of V-ATPases and their association with different
pathologies in vertebrates (11).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pac corresponds to a deletion between the two PacI restriction sites of A1E5. E5-lin-9 was
derived from E5-BK by SmaI and SacII restriction,
T4 DNA polymerase treatment, and religation. E5-BK*, introducing a
frameshift in exon 6, was derived from E5-BK by BamHI
restriction, Klenow treatment, and religation. The green fluorescent
protein (GFP) fusion in exon 4b, unc-32e4::GFP,
was constructed by introduction of a 3600-bp SmaI-PstI restriction fragment from E5-NS in the
HinDIII (blunted)-PstI-digested plasmid pPD95.81
(a gift from A. Fire).
-galactosidase in the pharynx by
5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside staining
(both pPD plasmids were gifts from A. Fire). Clones were injected into
e189 or f131 homozygotes and in
unc-32(e189 or f123 or f121)
dpy-17(e164)/qC1.
629 to 832, 394 to 1761, 1687 and
2162 to 3804, 2768 to 4622, and 4605 to 5851 (where position 1 is the A
of the initiator ATG) were fully sequenced on both strands. RNA was
extracted from the three alleles mutated in a splice junction,
e189, f120, and f131, and subjected to RT-PCR
analysis (see above) and sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The unc-32
uncoordinated mutant phenotype. Differential
interference contrast images of wild-type (A) and
unc-32(e189) mutant (B) worms as young adults are
shown. unc-32 mutants are characterized by a reverse ventral
coiler phenotype.
Complementation, dosage effects and molecular defects in unc-32 alleles
Pac and
E5-BK* constructs; see Fig. 2B). Although a rescue of the
zygotic lethality of f123 and f121 was obtained, the transformants were sterile (Table I and Fig. 2), possibly because
of an absence of germ line expression of the transgenes as previously
reported (23). Occasionally, the transforming plasmids rescued the
various phenotypes, Unc, lethal, maternal effect lethal, and defect in
gametogenesis, in a complex pattern. A particular transgenic array
could rescue the Unc but not the lethal phenotype, whereas another,
containing the same constructs, would do the opposite. This again
presumably reflects the complexity of the unc-32 locus (see
below).
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Fig. 2.
Characterization of the unc-32
gene. A, Genetic and physical maps of
the unc-32 region. The cosmids ZK637 and T05B2 were shown by
J. Sulston to rescue the uncoordinated phenotype of
unc-32(e189) mutants. B, top,
predicted and corrected genes in the indicated part of cosmid ZK637,
with invariant exons indicated by open boxes; alternative
exons in ZK637.8 are gray. The direction of transcription of
ZK637.7, which corresponds to lin-9 (35), and of ZK637.11 is
from right to left. For the other genes, it is
from left to right. The positions of the point
mutations of three unc-32 alleles are shown with
asterisks: e189 is a G-A transition in the
acceptor splice site in exon 4b; f131 is a G-A transition in
the donor splice site in exon 6; and f121 is a G-A
transition in exon 6. Bottom, inserts of the different
clones tested for their ability to rescue the different
unc-32 phenotypes. Let, lethal; Mel,
maternal effect lethal. Transformation rescue experiments were
performed in either e189, f131 or the lethal
alleles, and only a summary of the rescue is presented. For example,
the AIE5 construct rescued the movement and fertility
defects of f131 mutants but was associated with the
appearance of an f123-like maternal effect lethality such
that 85% of the eggs were dead. The clone
E5 Pac contains a deletion corresponding to the
major part of ZK637.8. The clone E5-BK* harbors a point
mutation resulting in a frame shift in the sixth exon and consequently
a premature stop codon, the position of which is marked by an
asterisk.
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Fig. 3.
The unc-32 gene
encodes several isoforms of V-ATPase a subunit.
The different unc-32 transcripts were determined by
sequencing different cDNA clones and RT-PCR products, and the
respective proteins are named as follows: UNC-32 A-F
contain, respectively, exons 4a/7b, 4b/7a, 4c/7b, 4a/7a, 4c/7a, and
4b/7b. Their GenBank accession numbers are, respectively, AF320899,
AF320900, AF320901, AF320902, AF320903, and AF320904. Their predicted
molecular masses, in kilodaltons, are, respectively, 103.4, 100.7, 102, 102.8, 101.3, and 101.1. Note that the different UNC-32 proteins differ
from both predicted ZK637.8a and 8b proteins. A, UNC-32 A,
F, and E protein sequences. Apart from the alternative exons (of
variable size, hence marked with dashes), the sequences are
identical and are not shown for the sake of clarity. Although the
fourth exons are not similar, exons 7b and 7a in the variants F and E,
respectively, are highly similar. Residues that are identical are
black; those that are similar are gray. The
vertical lines above the sequences mark the corresponding
exon-intron boundaries. B, exon-intron structure of the six
different alternative splice variants. The primers used for the RT-PCR
presented in C are also shown. Alternative exons are
gray. The relative abundances of different transcripts were
evaluated after digestion of RT-PCR products made from mRNA from
mixed stage wild-type worms and are shown as percentages.
C, different patterns of mRNA splicing are revealed by
RT-PCR in the unc-32 mutants, and their sequences are
presented. Top panel, analysis of the e189
mutation. On the left, the amplicon containing the fourth
exon was amplified with the primers 8.11 and 8.2 and digested with
HaeIII. Transcripts containing exon 4a (970 bp) or 4c (937 bp) are not cut, whereas transcripts containing exon 4b are cut in
three fragments (466, 407, and 49 bp; the 49-bp band is not visible in
this image). The e189 mutation removes a HaeIII
site, which is normally formed by the splicing of exon 3 to 4b, and
results in the appearance of a larger band (arrow) and the
loss of the 49-bp band (not shown). The band at 2375 bp results from direct PCR amplification of genomic DNA, shown in
lane gwt. On the right are presented
the sequences of both wild-type and e189 transcripts. The
e189 mutation removes the exon 4b acceptor splicing site; a
new acceptor splicing site, located 22 bp upstream of the normal one,
is used, introducing a stop codon. Bottom panel, analysis of
the f131 mutation. On the left, the amplicon
including the seventh exon was amplified with primers 8.5 and 8.6. Transcripts containing exon 7a (983 bp) or 7b (1001 bp) are not
resolved. The f131 mutation in the splice donor site of exon
6 provokes the apparition of a larger band (arrow). The
band at 2465 bp results from direct PCR
amplification of genomic DNA, shown in lane gwt.
On the right are presented the sequences of both wild-type
and f131 transcripts. The f131 mutation removes
the exon 6 donor splicing site; therefore, intron 6 is not spliced out
and fuses to exon 7a, resulting in the introduction of stop codons. The
bottom band is a mixture of various transcripts using
cryptic splice sites.
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Fig. 4.
Structure and sequence alignment of the four
C. elegans V-ATPase a
subunit genes. A, Intron-exon
structure of the four C. elegans V-ATPase a
subunit genes and the a1 and a3 human isoforms.
The open boxes represent exons drawn to scale, with the
exception of the largest ones. Note that the distance between the exons
is not to scale. The dotted lines show intron-exon
boundaries shared between two or more genes. B, amino acid
sequence alignment of the a subunit proteins from mouse and
nematode. Alignment of the three mouse a subunit protein
sequences (a1-a3; Ref. 9) and the four nematode sequences
(the UNC-32 A splice variant described here, VHA-5 (AAA81682), VHA-6
(CAA90758), and VHA-7, is shown. The latter is an edited version of the
Genefinder prediction (see "Experimental Procedures"). The position
of the nine putative transmembrane helices is indicated with
dotted lines, and the amino acids encoded by alternative
exons 4a and 7b are in italics.
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Fig. 5.
Evolutionary relationship between the
V-ATPase 100-kDa a subunit proteins in different
eukaryotes. The phylogenetic tree was constructed using the
C. elegans sequences shown in Fig. 4B, as well as
those of other organisms present in publicly accessible data bases. The
structure of the tree is compatible with an independent appearance of
different proteins subsequent to the evolutionary divergence of
vertebrates and invertebrates and reflects the fact that the UNC-32
protein is more closely related to the vertebrate a1 protein
than to other nematode proteins. Scale bar, 0.1 nucleotide
substitutions per site.
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Fig. 6.
Expression pattern of the four V-ATPase
a subunit genes in the nematode.
In situ hybridization images on embryonic, larval, and adult
worms with unc-32 (A-D), vha-5
(E-H), vha-6 (I-L), and
vha-7 (M-P) are shown. unc-32 is
expressed early and widely in the embryo (A); expression is
seen in the intestine (not shown), the gonad (arrow), and
some neurons in the head as well as motoneurons (arrowhead)
in the ventral cord in larvae (B and C) and adult
(D). vha-5 is also expressed early and widely in
the embryo (E); expression is seen in the excretory canal
(arrow) and some somatic gonad cells (arrowhead)
in larvae (F and G) and adult (H).
vha-6 is expressed only in the intestine, from the embryonic
coma stage (I) through larvae (J and
K) and adult (L). vha-7 is not
expressed in embryos and young larvae (M). Expression starts
at the L3-L4 larval stage (N) and is seen in early oocytes
(arrow) and in the spermateca (arrowhead) in L4
(O) and adult (P). Q, Confocal images
of an adult transgenic worm, carrying the construct
unc-32e4::GFP. Expression is limited to the
nervous system. mn, motoneurons.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Couillault, L. Guiraud and F. Jamen for their participation in this project, M. Craxton, A. Fire, and J. Kimble for the generous gift of clones and strains, I. Hope, A. Lynch, J. Sulston, and J. White for sharing unpublished results, C. Goridis and J.-F. Brunet for critical reading of the manuscript, and J. Thierry-Mieg, M. Weill, and J. Demaille for their support.
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FOOTNOTES |
---|
* This work was supported by institutional grants from Ministère de la Recherche et de l'Education Nationale, Institut National de la Santé et de la Recherche Médicale, and Centre National de la Recherche Scientifique.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF320899, AF320900, AF320901, AF320902, AF320903, and AF320904.
¶ To whom correspondence should be addressed: Laboratoire de Génétique et Physiologie du Développement, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de la Méditerranée, Luminy Case 907, 13288 Marseille Cedex 9, France. Tel.: 33-49126-9733; Fax: 33-49126-9726; E-mail: pujol@ibdm.univ-mrs.fr.
Present address: NCBI, NLM, National Institutes of Health,
Bethesda, MD 20894.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M009451200
2 J. Sulston, personal communication.
4 D. Thierry-Mieg and Y. Kohara, unpublished results.
5 A. Lynch and I. Hope, personal communication.
6 J. White and E. Southgate, personal communication.
3 GenBankTM accession numbers AAD34751, AAD34771, AAF53116, and AAF55550.
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ABBREVIATIONS |
---|
The abbreviations used are: V-ATPase, vacuolar-type H+-ATPase; bp, base pair; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SL, spliced leader; GFP, green fluorescent protein; Unc, uncoordinated.
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