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Mol Cell Biol, March 1998, p. 1553-1561, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Drosophila Gene for Antizyme
Requires Ribosomal Frameshifting for Expression and Contains an
Intronic Gene for snRNP Sm D3 on the Opposite Strand
Ivaylo P.
Ivanov,1
Karl
Simin,1
Anthea
Letsou,1
John F.
Atkins,1 and
Raymond
F.
Gesteland1,2,*
Department of Human Genetics1 and
Howard Hughes Medical Institute,2
University of Utah, Salt Lake City, Utah 84112
Received 20 October 1997/Returned for modification 14 November
1997/Accepted 18 November 1997
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ABSTRACT |
Previously, a Drosophila melanogaster sequence with
high homology to the sequence for mammalian antizyme (ornithine
decarboxylase antizyme) was reported. The present study shows that
homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5' open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that
encodes the carboxy-terminal three-quarters of the protein. Ribosomes
shift frame from the 5' ORF to ORF2 with an efficiency regulated by
polyamines. At least in mammals, this is part of an autoregulatory
circuit. The shift site and 23 of 25 of the flanking nucleotides which
are likely important for efficient frameshifting are identical to their
mammalian homologs. In the reverse orientation, within one of the
introns of the Drosophila antizyme gene, the gene for snRNP
Sm D3 is located. Previously, it was shown that two closely linked
P-element transposon insertions caused the gutfeeling
phenotype of embryonic lethality and aberrant neuronal and muscle cell
differentiation. The present work shows that defects in either snRNP Sm
D3 or antizyme, or both, are likely causes of the phenotype.
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INTRODUCTION |
The enzyme ornithine decarboxylase
(ODC) catalyzes the first step in the synthesis of polyamines and is
subject to extensive regulation (7). One mode of regulation
is modulation of its half-life. At least in mammals, the protein
antizyme (ODC antizyme) binds to, and inhibits, ODC (5, 8)
and then targets it for degradation by 26S proteasomes through a
ubiquitin-independent pathway (16, 25). Antizyme activity in
insect cells has recently been reported, and insect ODC has been shown
to be unstable and subject to destabilization by polyamines
(12). The isolation of a partial cDNA clone (17),
a genomic clone (22), and finally a full-length cDNA clone
(18) has been crucial for elucidating the mechanism of
antizyme synthesis. Surprisingly, translation of antizyme mRNA in at
least rat, human, and Xenopus requires a specific ribosomal
frameshift (9, 18, 28, 36). The amino-terminal portion is
encoded by open reading frame 1 (ORF1), and the remainder is encoded by
the overlapping ORF2 in the +1 reading frame. Programmed ribosomal
frameshifting in the overlap region produces a chimera from the two
ORFs. The frameshift efficiency is itself modulated by the
concentration of polyamines in cells, resulting in an autoregulatory
circuit (18, 28).
The mammalian antizyme frameshift occurs at the UCC serine codon
immediately before the UGA stop codon of ORF1, most likely by occlusion
of the U of UGA. A fraction of the ribosomes are switched to the +1
reading frame of ORF2 to complete synthesis of the ORF1-ORF2 product.
In addition to the UCCU shift site, three cis-acting RNA
elements contribute to the mammalian antizyme frameshift signal: the
UGA stop codon of ORF1, an RNA pseudoknot 3' of the shift site, and a
partially characterized signal nested within ~50 nucleotides (nt) 5'
of the ORF1 stop codon (18, 19).
Ribosomal frameshifting is an unusual but important mechanism of
translational control of gene expression (4, 6). It plays a
very important role in the expression of small genomes (viruses and
retrotransposable elements). The extent to which ribosomal
frameshifting is utilized in the expression of cellular genes is
largely unknown. The antizyme gene is a rare example of a eukaryotic
chromosomal gene regulated by translational frameshifting. Until now,
no evidence to suggest that expression of antizyme in any invertebrate
organism involves this kind of translational regulation has been
presented.
Interestingly, a homolog of antizyme ORF2 was discovered in a
Drosophila melanogaster cDNA ("guf cDNA"),
and its inactivation by P-element insertions was reported to lead to
embryonic lethality, deficiencies in terminal differentiation of
neuronal cells, and aberrant muscle development
the
gutfeeling phenotype (10, 30). On inspection of
the "guf cDNA" sequence, we had a gut feeling that this
interpretation was incorrect. By examining additional cDNA clones, we
discovered an alternative explanation for the gutfeeling
phenotype and deduced that there is a genuine D. melanogaster homolog of mammalian antizyme whose expression also
involves ribosomal frameshifting.
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MATERIALS AND METHODS |
DNA manipulations.
The oligonucleotide primers used were
GUTF1/S (5'-GCATCCGAATTCGGGCCTCTGTGGTGGTCC),
GUF/A2 (5'-CGTGCCCAAGCTTAGCTTCTCCTCGGCGAACTC), SNRNP/S2 (5'-GCATCCGAATTCAAGGGACTTGGTGGGACG),
GFEXN1/S
(5'-GCATCCGAATTCGACGACATCAGATCACAAATTGCTG), SP6UP/S (5'-GCATCCGAATTCTCAACTTTGGGACCTGCACC),
and SNRNP/A2
(5'-CGTGCCCAAGCTTGTGATTATGTGGCCCTCGGC). HindIII and EcoRI sites in all primers
are underlined.
HindIII and EcoRI sites were used to subclone
PCR products in pUC19. The region overlapping intron 2 of
guf1 was amplified with GUTF1/S and GUF/A2. The unknown
portion of intron 1 of guf1 was amplified with primers
SNRNP/S2 and GFEXN1/S. The 5' end of guf1 was amplified with
primers GUF/A2 and SP6UP/S (which is designed to prime sequences within
the cloning vector). The 5' end of guf2 was amplified with
primers SP6UP/S and SNRNP/A2. Southern blotting and other general
molecular biological techniques were done as described by Sambrook et
al. (31). D. melanogaster cDNAs were isolated
from a library constructed from 0- to 4-h embryos (2). D. melanogaster genomic clones were isolated from a cosmid
library constructed from the iso-1 fly stock (35). It should
be noted that the probe chosen by Salzberg et al. (30) to
screen for guf cDNA clones could not have identified spliced
guf1 (encoding antizyme) because it corresponds to a region
inside intron 1 of that message; however, it could have identified
guf2 (encoding small nuclear ribonucleoprotein [snRNP] Sm
D3) clones.
In vitro transcription and translation.
CsCl-purified DNA
templates were digested with EcoRI (NE and GUF-B) and
BglII (GUF-B). One microgram of restricted DNA was used as
the template for in vitro transcription with phage T7 or SP6 RNA
polymerase (Promega) for NE or GUF-B, respectively, according to the
manufacturer's recommendations. After transcription, the templates
were destroyed by incubation with RNase-free DNase (Promega) and the
RNAs were recovered by phenol-chloroform extraction and ethanol
precipitation. Transcripts were resuspended in 20 µl of 5 mM
dithiothreitol containing 20 U of RNasin (Promega). One microliter of
RNA was used to program a translation reaction with 10 µl of wheat
germ (Promega). Then 50 mM potassium acetate was added to the reaction
mixtures for optimal translation. The endogenous level of spermidine in
the extract was 0.5 mM. Reaction mixtures were supplemented with
spermidine, as indicated in the figure legends.
[35S]methionine-labeled protein products were separated
by electrophoresis through 16% polyacrylamide Tris-Tricine gels
(Novex). Gels were fixed, dried under vacuum, and exposed to X-ray
film. ORF1 and ORF2 for fly antizyme each contain three methionine
codons. ORF1 and ORF2 for rat antizyme contain two and one methionine
codons, respectively.
Northern blot analyses.
Total RNA was extracted from 0- to
24-h Canton S embryos by a standard boiling-phenol method. Twenty-five
milligrams of RNA per lane was electrophoresed on a 1.2% agarose gel
containing formaldehyde. Transcript sizes were determined relative to a
0.24- to 9.5-kb RNA ladder (Gibco BRL) and visualized by ethidium
bromide staining. The RNA was transferred by capillary transfer to a
GeneScreen Plus nylon membrane (DuPont) and cross-linked with UV light
(0.12 J/cm2). [32P]dCTP-labeled antisense DNA
probes were generated with a Prime-It II labeling kit (Stratagene),
replacing the random primers with 25 ng of antisense primers.
Exon-specific DNA templates were generated by PCR (guf1 exon
2 and 3 [and intron 2] genomic DNA, guf2 exon 1, and
guf1 exon 1). Filters were hybridized in 50% formamide at
42°C and then washed at high stringency.
The Northern blot analysis presented by Salzberg et al. (
30)
appears to contradict our results. In their analysis, a 6-kb
PstI genomic fragment spanning the
gutfeeling
locus (including
both transcripts described in this paper) was used as
a probe.
Major (2,100-nt) and minor (nonreproducible) (1,600-nt)
transcripts
were observed. We can provide an explanation for the
discrepancy
between the observed RNA sizes. The sizes of the RNA
markers in
lane 1 on the gel presented in Fig. 6 of reference
30 appear
to be incorrect. The best indication of
this comes from analysis
of the RNA band corresponding to ribosomal
protein 49 (
rp49).
According to the size markers on their
gel,
rp49 mRNA has a size
of ~1,400 nt. The real size of
rp49 mRNA is ~600 nt (
26). When
this is taken
into consideration, the Northern blot presented
by Salzberg et al.
(
30) is in good agreement with our findings,
with the
abundant 2,100-nt RNA corresponding to the antizyme mRNA
and the
1,600-nt nonreproducible RNA corresponding to the snRNP
Sm D3 mRNA.
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RESULTS |
Translational frameshifting is required for the expression of a
D. melanogaster homolog of antizyme.
A computer search
with the BLAST algorithm (1) for sequences similar to the
mammalian antizyme frameshift site revealed a stretch of 28 nt in the
5' untranslated region (UTR) of the "guf cDNA." In this
sequence, 26 nucleotides were identical to those that define the rat
antizyme frameshift site (Fig. 1). The high degree of similarity is significant, since previous in vitro experiments suggested that such a sequence could stimulate measurable levels of ribosomal frameshifting (18) in mammals. We
speculated that the presence of a potential frameshift site on the same
cDNA molecule that is predicted to encode a protein with a significant homology to mammalian (and indeed all known vertebrate) antizymes was
more than coincidental. The homology at a potential frameshift site is
in addition to the seemingly disconnected downstream region of homology
discovered previously (30). The reported 5' UTR of the
"guf cDNA" is unusually large (about 1,250 nt) and
contains 23 AUG codons (Fig. 2C). These
features led us to hypothesize that the reported guf cDNA
clone represents an unspliced D. melanogaster antizyme clone
with an intronic sequence which, when spliced out, would unite the two
homologous regions of the antizyme sequences.
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FIG. 1.
Nucleotide sequence comparison of antizyme frameshift
sites. Black shading indicates identity among at least three sequences.
Boxes with standard letters indicate identity within the most conserved
region among all four sequences. Sequences involved in the formation of
the UGA stop codon of ORF1 are indicated by a box with boldface
letters. The stems of the vertebrate 3' pseudoknot are underlined.
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FIG. 2.
Schematic representation of the gutfeeling
locus. The sequence similar to the mammalian antizyme frameshift site
is indicated by a red box. Panels A to D are to the same scale, and
panels A to C are positioned relative to each other. (A) D. melanogaster antizyme mRNA (guf1). Full bars represent
stop codons; half bars represent AUG codons. (B) The transcriptional
unit of D. melanogaster antizyme relative to guf
cDNA. (C) ORF map of "guf cDNA" as defined previously
(30). Green half bars designate AUG codons in the 5' UTR.
The green box indicates the gutfeeling ORF proposed
previously (30). Red full bars indicate stop codons in the
+1 frame situated between the region similar to the mammalian antizyme
frameshift site and the downstream ORF whose product exhibits amino
acid homology to vertebrate antizyme. The blue box indicates a region
with high homology to sequences for S. cerevisiae and human
snRNP Sm D3. Newly identified splice sites are indicated by arrows: red
arrow, 5'; blue arrow, 3'. The positions of the two P-element
insertions causing the gutfeeling phenotype (30)
are shown by black arrows. (D) D. melanogaster snRNP Sm D3
transcriptional unit relative to "guf cDNA." (E)
Physical map of the gutfeeling locus. The exons of the
antizyme mRNA are represented by green bars. The aberrant exon 1 of
GUF-A is shown in orange. The exons of snRNP Sm D3 are represented by
blue lines. Intervening sequences are in red. The positions of the two
primers used in determining the size of intron 1 of the antizyme
sequence are indicated by red arrows. P, PstI; S,
SalI.
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To search for the predicted intron, the region was amplified with PCR
primers on either side. The sense primer corresponded
to the
guf cDNA sequence homologous to the antizyme frameshift
site. The antisense primer corresponded to the
guf cDNA
sequence
showing the highest amino acid homology to antizyme (see
Materials
and Methods). The PCR product obtained by amplifying genomic
DNA
was subcloned and sequenced. The sequence corresponded perfectly
with the published "
guf cDNA" sequence. In contrast, the
PCR product
from an embryonic (0 to 4 h)
D. melanogaster cDNA library was
shorter than the genomic PCR product
(data not shown). We did
not find a cDNA PCR product with the same size
as the genomic
PCR product, as would be expected from the reported
"
guf cDNA"
sequence which is completely colinear with
the genomic sequence.
The sequence of the cDNA PCR product revealed the
absence of 66
nt compared to the genomic sequence. The ends of the
missing sequence
have similarity to
D. melanogaster 5' and
3' splice site consensus
sequences (
24) (Fig.
3A). Splicing out this intron (later
shown
to be intron 2) removes, as predicted, the four in-frame stop
codons (frame 2 on Fig.
2C) between the sequence homologous to
the
mammalian antizyme frameshift site and the downstream sequences
which
are homologous to the equivalent parts of mammalian antizyme
mRNA. The
intron removal provides potential accessibility to these
downstream
sequences via a frameshifting mechanism. Furthermore,
splicing removes
the AUG translation initiation codon of
guf cDNA
proposed
previously (
30) (the next in-frame AUG is well past
the
point where the homology between
guf and the antizyme
sequence
begins). This suggests that the initiation codon utilized is
present
in a different ORF.
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FIG. 3.
Homology between exon-intron boundaries of the newly
identified antizyme and snRNP Sm D3 transcripts and the consensus
sequences for 5' and 3' splice sites of D. melanogaster. The
invariant GU (5' splice site) and AG (3' splice site) are in boldface
and underlined or overlined. The point of splicing is indicated by an
arrow. The ratio of identical nucleotides to total nucleotides is also
given.
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The antizyme sequence was also amplified from
Drosophila
virilis genomic DNA with the same set of PCR primers. Sequencing
revealed a high degree of homology between the antizyme sequences
of
the two flies (data not shown). The only region lacking homology
corresponds to the newly discovered antizyme intron. In fact,
the
putative antizyme intron of
D. virilis has a different size
(69 nt) but has potential 5' and 3' consensus splice sites (Fig.
3C).
cDNA clones were isolated from the same embryonic
D. melanogaster library by hybridization to probes made from the PCR
products
described above. Two clones, GUF-A and GUF-B, were chosen for
sequencing. The 5' end of GUF-B contained an exon (exon 1) not
reported
in the
guf cDNA analysis reported previously
(
30).
From the 3' end of exon 1, the sequence jumps to nt
+1015 of the
published
guf cDNA sequence (Fig.
2B and C).
The sequence surrounding
nt +1015 corresponds well to the consensus 3'
splice site (Fig.
3B). The only other intron we found that is removed
to give GUF-B
corresponds to the intron described in the preceding
paragraph.
This conclusion is supported by PCR analysis (data not
shown).
The first AUG codon of GUF-B initiates an ORF (ORF1) ending
with
the UGA stop codon of the sequence which is homologous to the
vertebrate antizyme frameshift site (Fig.
2A). Sequences downstream
define a second ORF (ORF2), homologous to the vertebrate antizyme
gene.
The two ORFs in GUF-B overlap such that they would be fused
by a +1
translational frameshift, analogous to the genes encoding
mammalian
antizyme. The transcript represented by GUF-B is called
guf1.
The unique 5' exon in GUF-B did not correspond to any sequence in the
guf cDNA, and it had no homology to vertebrate antizyme
sequences. Consistent with the hypothesis that exon 1 is part
of the
D. melanogaster antizyme gene, all three exons were
recovered
on a single cosmid clone (data not shown). Additional mapping
revealed that all three exons were contained in a single 8.2-kb
HindIII fragment. Using primers corresponding to
antizyme sequences
in exon 1 and intron 1 (Fig.
2E), we determined that
intron 1
is ~6.2 kb in length. To further show that exon 1 corresponds
to the 5' end of the
D. melanogaster antizyme
mRNA transcript,
the 5' end of antizyme message was amplified from the
0- to 4-h-embryo
cDNA library with primers within the cloning vector
and exon 3.
The predominant product was subcloned and sequenced.
Sequence
analysis of this PCR product demonstrated that
D. melanogaster antizyme cDNAs contain 5' ends beginning with exon 1 of GUF-B.
GUF-A has an unusual 5' end. Its 5'-most sequence corresponds to nt
+133 of the previously published
guf cDNA. From there,
GUF-A
and the published sequence are colinear until nt +356 of
the latter,
corresponding to exon 1 of GUF-A (Fig.
2E). The other
two exons of
GUF-A are similar to exons 2 and 3 of GUF-B. Exon
1 of GUF-A contains
numerous AUGs followed by a number of stop
codons in all reading
frames. This finding, combined with our
analyses showing that GUF-B
contains the predominant exon 1 of
guf1, led us to conclude
that GUF-A is an aberrant transcript
of
D. melanogaster
antizyme.
The 3' end of GUF-B is physically close to the previously reported 3'
end of
guf cDNA. GUF-A has an additional ~350 nt at
its 3'
end that are not present in GUF-B. PCR and partial sequencing
analysis
(data not shown) indicate that the
guf1 transcript also
contains these additional 350 nt. The 3' end of GUF-B corresponds
to a
sequence within GUF-A containing 17 consecutive A's (Fig.
4A), which would provide a good template
for the poly(dT) primer
used in generating the cDNA library. It appears
that the 3' end
of GUF-B is a cDNA artifact. Interestingly, the 3' end
of the
published "
guf cDNA" sequence corresponds to a
stretch of 24 nt,
present in both GUF-B and GUF-A, that contains 20 A's (Fig.
4A).
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FIG. 4.
(A) Nucleotide sequence and predicted amino acid
sequence of D. melanogaster antizyme (guf1). The
predicted amino acid sequences for both ORF1 and ORF2 are given. The
UGA stop codon of ORF1 is boxed. The most likely position of
translational frameshifting is indicated by a diagonal arrow. The two
A-rich regions (discussed in the text) within the 3' UTR are
underlined. The 3' end of "guf cDNA" in relation to the
nucleotide sequence presented here is indicated by a small vertical
arrow. (B) Comparison between the amino acid sequences of D. melanogaster and D. virilis (partial) antizyme proteins
and their frog, rat, and human counterparts. A black background
indicates amino acid identities among at least three proteins.
Boldfacing indicates amino acid similarities among at least four
proteins. A symbol following the designation for D. melanogaster, D. virilis, X. laevis, or
Rattus rattus indicates a PEST sequence, and the identity of
the amino acids involved is indicated by the position of the
corresponding symbol over the top line of that set of sequences.
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ORF1 of
guf1 encodes a polypeptide of 61 amino acids. By
comparison, rat and
Xenopus antizyme ORF1s encode
polypeptides of
68 (most likely) (
18) and 58 (
9)
amino acids, respectively,
but amino acid homology between
D. melanogaster and vertebrate
antizyme proteins is limited and is
concentrated near the C termini
of the ORF1-encoded polypeptides (Fig.
4B).
If expression of
D. melanogaster antizyme is similar to that
of vertebrate antizyme proteins (see Discussion), +1 ribosomal
frameshifting at a UCC serine codon immediately preceding the
UGA stop
codon of
guf1 ORF1 would lead to translational fusion
of
ORF1 and ORF2 of
guf1 to produce a polypeptide of 254 amino
acids (Fig.
4A). This expected polypeptide is acidic (predicted
pI = 4.78), with a predicted molecular mass of 28,282 Da. A computer
search with the PROSITE algorithm identified several potential
sites
for posttranslational modifications, including five protein
kinase C
and nine casein kinase II phosphorylation sites. Seventeen
amino acids
encoded by ORF2 link the ORF1 polypeptide to the last
176 amino acids
of the proposed
gutfeeling protein. The homology
of this
region to vertebrate antizyme is shown in Fig.
4B. The
first 64 amino
acids encoded by ORF2 have no apparent homology
to vertebrate antizyme
and are unusual in that they contain 16
(25%) serine residues. The
same region is also present in
D. virilis antizyme but
contains even more serines, i.e., 19. A sequence
pattern termed PEST by
its discoverers (
27) occurs within this
region of
D. melanogaster antizyme and contains most of the serines
(Fig.
4B).
There is experimental evidence in several proteins
that PEST sequences
confer lability, though this is as yet unproven.
The scores with the
PEST algorithm for the
D. melanogaster and
D. virilis sequences in question are 6.8 and 21.4, respectively.
Frameshifting in decoding
guf1 was tested in a wheat germ in
vitro translational system with RNA transcribed from GUF-B digested
with
EcoRI. As a control, a rat antizyme transcript was also
translated.
The major large (>10-kDa) product from translation of
guf1 had
an apparent molecular mass of 28 kDa (Fig.
5), close to the predicted
molecular mass
(28.3 kDa) of an ORF1-ORF2 fusion (the origin of
a minor protein
product ~6 kDa longer than the major product is
unknown). The major
large product from translation of rat antizyme
RNA had an apparent
molecular mass of 25 kDa (predicted molecular
mass = 25.2 kDa)
(Fig.
5A). To confirm that the 28-kDa protein
was indeed the product of
transframe translation of ORF1-ORF2,
a transcript from GUF-B digested
with
BglII (which has a unique
site within ORF2) was
translated in a wheat germ extract. The
size of the major large product
was reduced to an apparent molecular
mass of 15 kDa, as expected
(predicted molecular mass, 14.7 kDa)
(Fig.
5B). The large number (at
least four) of small (<10-kDa)
products on the gels (Fig.
5) makes it
difficult to discern the
termination product of ORF1 (predicted
molecular masses of 6.7
kDa for fly and 7.4 kDa for rat).
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FIG. 5.
In vitro translation of D. melanogaster
antizyme in wheat germ extracts. Protein markers are on the left in
both panels. (A) Rat and Drosophila (GUF-B) antizymes
synthesized in the presence of increasing concentrations of spermidine
(Spd) (0.5 to 1 mM) with products separated on a 16% Tricine gel. The
products most likely to be from translational termination at the stop
codon of ORF1 of rat and fly antizymes (Az) are indicated by arrows.
(B) RNAs transcribed from GUF-B digested with EcoRI (cutting
3' of the cloned cDNA) and BglII (cutting inside ORF2)
translated in the presence of increasing concentrations of spermidine.
Translation of brome mosaic virus (BMV) RNA was a control.
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Addition of the polyamine spermidine increased synthesis of the 28-kDa
protein from
guf1 (Fig.
5). As expected (
18,
28),
a similar induction was seen for the rat 25-kDa antizyme protein
(note
that even though its absolute amount is reduced, its abundance
relative
to the smaller products on the gel is increased). Comparison
of the
products after stimulation with spermidine strongly suggests
that the
frameshift efficiency (in the in vitro heterologous system)
of
guf1 is comparable to, or greater than, that of the rat
antizyme
sequence in reticulocyte lysates, which is as high as 19%
(
18).
Translation of
guf1 mRNA in a rabbit
reticulocyte lysate gave
very similar results (data not shown), but the
ubiquitous hemoglobin
protein precluded visualization of the ORF1
termination products.
D. melanogaster homolog of the snRNP Sm D3 gene present
in the gutfeeling locus.
A substantial portion of the
5' UTR of the guf cDNA has a very strong homology, in the
antisense orientation, to human and Saccharomyces cerevisiae
snRNP Sm D3 genes (Fig. 2C), suggesting that there could be a second
gene within the gutfeeling locus. To investigate this
possibility, an early embryonic library was screened for cDNA clones by
hybridization with a probe corresponding to the region of the published
guf cDNA sequence with the highest homology to snRNP Sm D3
genes from humans and yeast. Two clones (GUF2-1 and GUF2-3) were
sequenced. Both clones contained transcribed sequence in the antisense
orientation relative to the guf1 transcript (Fig. 2D),
indicating that they represented a distinct transcriptional unit,
designated guf2. The two sequenced clones were almost
identical, one having an additional 4 nt at its 5' end. To determine if
GUF2-1 and GUF2-3 are representative of the 5' end of the
guf2, the 5' region of this mRNA was amplified (from a 0- to
4-h-embryo cDNA library), subcloned, and sequenced. The PCR clone
contained several additional nucleotides at its 5' end, compared to the
cDNA clones. This data was used to define nt 1 of guf2. The
first nucleotide of the guf2 transcript corresponds to nt
+495 of the previously published guf cDNA sequence. The 5'
UTR of guf2 is 151 nt long. The two P-element insertions
that cause the gutfeeling phenotype map to nt +28 and +47 of
the 5' UTR of guf2. Exon 1 of guf2 is 472 nt
long. PCR analyses indicated that the remainder of guf2 is
derived from a single exon (exon 2) and that the intervening sequence
(intron 1) has a size of ~70 nt (data not shown). guf2 cDNAs contain a single long ORF encoding a protein of 151 amino acids
(Fig. 6A). The predicted protein is basic
(predicted pI = 10.55) and has a molecular mass of 15,582 Da. A
computer search with the BLAST algorithm revealed very high homology
with snRNP Sm D3 of Homo sapiens and S. cerevisiae (Fig. 6B). The highest homology is within the first 90 amino acids of the guf2 protein (close to the size of the
S. cerevisiae protein). The C-terminal half of the
guf2 protein contains an arginine-glycine (R-G)-rich sequence. Similar (but shorter) R-G-rich regions are present in the C
termini of human snRNP Sm D3 and snRNP Sm D1 proteins (15). Interestingly, the guf2 sequence homologous to the yeast
snRNP Sm D3 sequence is located in exon 1, while the sequence encoding the R-G-rich domain is located mostly in exon 2.
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FIG. 6.
(A) Nucleotide sequence and predicted amino acid
sequence of D. melanogaster snRNP Sm D3. (B) Sequence
alignment between D. melanogaster snRNP Sm D3 protein and
snRNP Sm D3 from H. sapiens and S. cerevisiae.
Dark background indicates amino acid identities between at least two
proteins. Boldfacing indicates amino acid similarities between at least
two proteins.
|
|
Expression analysis of guf1 and guf2
transcripts.
cDNA analyses led us to predict two transcripts
within the gutfeeling locus, one of 750 nt (guf2)
and the other of 1,650 nt (guf1). Northern blot analysis was
performed with probes for each of the two transcripts (guf1
and guf2). The probe for guf1 (exon 1 or exons 2 and 3) revealed an RNA of 1,800 nt (Fig.
7, lanes 1 and 3). The probe for
guf2 (exon 1) revealed a single RNA species with an apparent
size of 950 nt (Fig. 7, lane 2). The sizes corresponded well to the
deduced sizes of the two transcripts [allowing for 150 to 200 nt of
poly(A) tails]. A Northern blot study presented by Salzberg et al.
(30) appears to disagree with our analysis. However, upon
more careful consideration, the two sets of data are in good agreement
(for details see Materials and Methods).
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 7.
Northern blot analysis of guf1 and
guf2 transcripts. Shown is a Northern blot of embryonic (0- to 24-h) RNA probed with DNA fragments corresponding to exons (Ex) 2 and 3 of the antizyme sequence (Az) (guf1) (lane 1), exon 1 of the snRNP Sm D3 sequence (guf2) (lane 2), and exon 1 of
the antizyme sequence (lane 3).
|
|
The
guf1 transcript is quite abundant. While screening the
D. melanogaster cDNA library for antizyme cDNA clones, we
found
a multitude of positive clones, at least 2 orders of magnitude
more than for
guf2. In addition, during the writing of this
paper,
the
D. melanogaster expressed sequence tag sequencing
project
generated no fewer than 18 clones (from a 0- to 24-h-embryo
cDNA
library) corresponding to the
guf1 transcript. High
levels of
antizyme message in mammalian tissues were also reported
(
17).
No clones corresponding to the previously published
"
guf cDNA"
clone or to
guf2 were present in
the data bank.
| |
DISCUSSION |
Antizyme in Drosophila: utilization of programmed
frameshifting.
The discovery of intron 2 (in guf1)
demonstrated that the presence of two regions of guf cDNA
with homology to mammalian antizyme cDNA is not merely coincidental.
Splicing out intron 2 removes the only potential start codon for
independent initiation of ORF2. It also removes the blocking in-frame
stop codons to leave unencumbered overlapping ORF1 and ORF2. This
permits a major expansion to the theory that this mRNA encodes a
homolog of mammalian antizyme. Evidence that this sequence causes
efficient regulated programmed frameshifting directly comparable to
that of its mammalian counterpart is provided by the in vitro
translation experiments which yielded a transframe protein of the
expected size that was responsive to polyamine concentration. These
results, combined with our knowledge of the expression of mammalian
antizyme, lead us to conclude that the expression of D. melanogaster antizyme involves programmed, polyamine-regulated,
translational frameshifting.
Programmed frameshifting is known to occur in single-celled eukaryotes
(yeast and protozoa) and in
Xenopus but has not previously
been discovered in any intermediate organism. The finding of antizyme
programmed frameshifting in
Drosophila provides the
opportunity
to study the evolution of this recoding event, which
involves
a transitory alteration of the rules of readout of the genetic
code. Studies on this evolutionary aspect will be reported elsewhere,
but the degree of conservation of shift site sequences is considered
below. Notably, however, the 3' mRNA message feature that is an
important stimulator, a pseudoknot, for the recoding signal in
mammalian systems is not recognizable in
Drosophila, and the
identification
of its presumed alternative is of major interest.
Of the three known RNA elements that stimulate antizyme frameshifting
in mammalian cells, the other two are present in
guf1.
One
of these is the UGA stop codon of ORF1. It is interesting
that even
though in vitro translation experiments have shown that
the other two
stop codons (UAG and UAA) are almost as effective
in stimulating
frameshifting in decoding mammalian antizyme sequences
(
18),
so far all eukaryotic antizyme genes have UGA as the stop
codon of
ORF1. Another stimulatory element, a sequence immediately
5' of the UGA
stop codon, is also likely to be present in the
D. melanogaster antizyme sequence. Of 18 nucleotides 5' of the
UGA
stop codon, 16 are identical for
guf1 and the rat antizyme
sequence (with one of the two mismatches being an antizyme
polymorphism)
(Fig.
1). This level of 5' sequence homology is striking
and provides
a clear indication that this region plays an important
role in
antizyme frameshifting. The apparent absence of an RNA
pseudoknot
3' of the UGA stop codon of
guf1 is puzzling. The
nucleotides
within the stems of this pseudoknot are absolutely
conserved among
all known vertebrate antizyme sequences (Fig.
1 and
unpublished
data). Despite the apparent absence of a 3' pseudoknot,
there
is nucleotide sequence homology between this region of
guf1 and
vertebrate antizyme sequences (Fig.
1). Perhaps
some other RNA
structure is present in this region of
guf1.
Indirect evidence
supports such a hypothesis. Comparison of
D. melanogaster and
D. virilis antizyme nucleotide
sequences reveals that the 53 nt
between the UGA of the putative
frameshift site and intron 2 (a
region most likely to contain a
stimulatory 3' RNA structure)
are completely conserved between the two
species, even though
the conservation of the rest of the known exonic
sequences is
only 75%. Computer programs predict several stem loops in
this
region, but the relevance of any of them to translational
frameshifting
is unknown. Even though additional frameshift-stimulatory
elements
in the
Drosophila antizyme sequence are not
obvious, there is
a strong indication that there is more to the
guf1 frameshift
site than the 28-nt region homologous to the
mammalian antizyme
frameshift site. Results presented previously
(
18,
19) indicate
that the 28 nt alone cannot stimulate more
than 3% frameshifting
in vitro. Our data for the in vitro translation
of
guf1 strongly
suggests that the frameshift efficiency in
that system is much
higher than 3%, thus implying the existence of
additional frameshift-stimulatory
signals in
Drosophila
antizyme mRNA.
Rat antizyme is a short-lived protein (
8). It contains a
PEST sequence associated with proteins that rapidly turn over
(
27).
Xenopus and rat antizymes also contain PEST
sequences
(
9) (Fig.
4B). The predicted
Drosophila
antizyme protein contains
a PEST sequence within a region with no
apparent homology to vertebrate
antizymes (Fig.
4B). It should be noted
that the PEST sequences
of rat,
Xenopus, and
Drosophila antizymes are located in different
regions of the
protein, and so their significance is not clear.
However, the presence
of the PEST sequence may indicate that
Drosophila antizyme,
like rat antizyme, is also a short-lived protein.
snRNP Sm D3 gene of Drosophila: a nested gene.
Our
analysis revealed a second gene in the gutfeeling locus.
This gene has a transcriptional unit (guf2) that is oriented in a direction opposite to that of the transcriptional unit
guf1. This second gene, with two exons, is entirely within
intron 1 of guf1 (Fig. 2E). This nested gene organization,
where one gene exists within an intron of another gene (on the opposite
strand), is unusual; however, several such examples have been described (reference 21 and references therein). This gene
encodes a protein that has very high homology to snRNP Sm D3 proteins
from other organisms. snRNPs U1, U2, U4/U6, and U5 are essential for
pre-mRNA splicing (23, 34). Two classes of snRNP proteins
exist. The class Sm includes proteins common to all four snRNPs. The
other class includes proteins that are specific to each snRNP particle. snRNP Sm D3 is one of the proteins common to all snRNPs
(14). On the basis of the amino acid homology (Fig. 6B), we
conclude that the guf2 product is the same as D. melanogaster snRNP Sm D3.
R-G-rich regions.
The C terminus of D. melanogaster
snRNP Sm D3 contains an R-G-rich region (Fig. 6). A search revealed a
number of proteins in the public data bank that contain R-G-rich
regions, including fibrillarin (Schizosaccharomyces pombe),
NAB2 (S. cerevisiae), FMR-1 (H. sapiens), GAR1
(S. cerevisiae), EWS (H. sapiens), nucleolin (Xenopus laevis), heterogeneous ribonuclear particle protein
A1.b (X. laevis), glycine-rich RNA-binding protein GRP1A
(Sinapis alba), GAM1 (S. cerevisiae), basic
fibroblast growth factor (Rattus norvegicum), Epstein-Barr
virus nuclear antigens 1 and 2 (H. sapiens), and ALY
(Mus musculus). The role of the R-G-rich sequences in these proteins is not entirely clear. Many, but not all, of these R-G-rich regions contain the RGG RNA binding box (3, 11). It is
thought that RGG box regions bind to RNA through a
non-sequence-specific mechanism. Even though snRNP Sm D3 from
Drosophila contains two RGG repeats, the human homolog
contains none in its R-G-rich region. In fact, not all proteins we have
identified that contain R-G-rich regions are thought to bind RNA (some
are transcription factors with no known RNA binding motifs).
The only feature common to all these proteins that contain R-G-rich
sequences is that they all are located in the nucleus.
This raises the
possibility that, at least in some proteins, the
R-G-rich region could
be a nuclear localization sequence (NLS)
or be involved in some aspect
of nuclear localization (for nucleolin
and NLS-binding protein (NSR1),
regions other than the R-G-rich
one have been implicated as NLSs
[
32,
37]). The feature that
unites all NLSs is their
richness in basic amino acids (
33),
and the R-G-rich region
would most likely satisfy this criterion.
The presence of an RNA
binding motif and NLS in the same region
of a protein is not
unprecedented. Previously published work (
13)
has shown that
for the majority of nuclear proteins for which
an NLS and DNA- or
RNA-binding domain have been determined, the
two are either overlapping
or flanking. Perhaps the best indication
that the R-G-rich region might
be involved in nuclear localization
comes from an analysis of
fibrillarin proteins from different
species. Fibrillarin is needed for
pre-rRNA processing and is
located in the nucleus (
20). All
known fibrillarins from eukaryotic
species contain an R-G-rich region
in their N termini. A genuine
homolog of fibrillarin appears to exist
in several
Methanococcus (archaeon) species (GenBank
accession no.
X73987,
X73988,
and 2127901). As our hypothesis predicts,
archaeon fibrillarin
proteins (which do not have to be transported
across a nuclear
membrane) do not have the R-G-rich region, even though
they have
extensive similarity to the rest of their eukaryotic
homologs.
gutfeeling.
Since both P-element insertions responsible
for the gutfeeling mutant phenotype map to the 5' UTR of
D. melanogaster snRNP Sm D3 mRNA and therefore would cause a
severe gene disruption, we propose that defective expression of snRNP
Sm D3 is a likely contributor to the gutfeeling phenotype.
However, since both P elements are also in intron 1 of the antizyme
gene homolog, they could disrupt its splicing or possibly its
transcription and so contribute to the phenotype. Perhaps snRNP Sm D3
and antizyme contribute to different aspects of the
gutfeeling phenotype. This could be determined by
identifying and analyzing point mutations in each of the genes for the
two proteins.
Knockout experiments have demonstrated that the snRNP Sm D3 gene is an
essential gene in
S. cerevisiae (
29). It is
reasonable
to assume that the same is true for
D. melanogaster. How disruption
of snRNP Sm D3, a protein which is an
integral part of eukaryotic
spliceosomes, would result in specific
deficiencies of neuron
and/or muscle differentiation is not clear.
Whatever the mechanism,
it is most likely nonspecific. For example, it
is possible that
mutations disrupting zygotic snRNP Sm D3 function
coupled with
gradual dilution of the pool of maternally supplied
protein would
initially and differentially affect the splicing of a
subset of
mRNAs, one or more of which are required for neuron and/or
muscle
cell differentiation. The possible role of antizyme in the
gutfeeling phenotype has already been discussed at length
previously (
30).
The discovery that
Drosophila antizyme is apparently
regulated by translational frameshifting suggests that this regulatory
event is much more common than previously thought. It is in fact
very
likely that translational frameshifting is involved in the
regulation of antizyme in all animals expressing this protein.
| |
ACKNOWLEDGMENTS |
We thank Norma Wills for kind help with the translation
experiments and Senya Matsufuji for discussions.
K.S. is a Developmental Biology Training Grant (5T32HD07491-03)
recipient. A.L. has a JFRA-657 award. R.F.G. is an investigator from
the Howard Hughes Medical Institute. This work was also supported by a
grant (RO1-GM48152) from the National Institutes of Health to J.F.A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Human Genetics, The University of Utah, 15N, 2030 E, Room 6160, Salt Lake City, UT 84112-5330. Phone: (801) 581-5190. Fax: (801) 585-3910. E-mail: rayg{at}howard.genetics.utah.edu.
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Abstract
Introduction
