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Molecular and Cellular Biology, November 2000, p. 8198-8208, Vol. 20, No. 21
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Arginine-Rich Regions Mediate the RNA Binding and
Regulatory Activities of the Protein Encoded by the
Drosophila melanogaster suppressor of sable Gene
Michael A.
Turnage,
Paul
Brewer-Jensen,
Wen-Li
Bai, and
Lillie L.
Searles*
Department of Biology and Curriculum in
Genetics and Molecular Biology, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599-3280
Received 30 May 2000/Returned for modification 11 July
2000/Accepted 1 August 2000
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ABSTRACT |
The Drosophila melanogaster suppressor of sable gene,
su(s), encodes a novel, 150-kDa nuclear RNA binding
protein, SU(S), that negatively regulates RNA accumulation from mutant
alleles of other genes that have transposon insertions in the 5'
transcribed region. In this study, we delineated the RNA binding domain
of SU(S) and evaluated its relevance to SU(S) function in vivo. As a
result, we have defined two arginine-rich motifs (ARM1 and ARM2) that
mediate the RNA binding activity of SU(S). ARM1 is required for in
vitro high-affinity binding of SU(S) to small RNAs that were previously
isolated by SELEX (binding site selection assay) and that contain a
common consensus sequence. ARM1 is also required for the association of
SU(S) with larval polytene chromosomes in vivo. ARM2 promotes binding
of SU(S) to SELEX RNAs that lack the consensus sequence and apparently
is neither necessary nor sufficient for the stable polytene chromosome
association of SU(S). Use of the GAL4/UAS system to drive ectopic
expression of su(s) cDNA transgenes revealed two previously
unknown properties of SU(S). First, overexpression of SU(S) is lethal.
Second, SU(S) negatively regulates expression of su(s)
intronless cDNA transgenes, and the ARMs are required for this effect.
Considering these and previous results, we propose that SU(S) binds to
the 5' region of nascent transcripts and inhibits RNA production in a
manner that can be overcome by splicing complex assembly.
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INTRODUCTION |
Eukaryotic protein-coding RNAs are
typically transcribed as larger pre-mRNAs that are processed to a
mature form. Pre-mRNA processing is coupled to transcription (7,
8, 27, 42) and involves a complex set of events including the
addition of a 7-methylguanosine cap to the 5' end, splicing to remove
internal introns, and cleavage/polyadenylation of the 3' end.
Interactions between the cellular transcription and RNA processing
apparatuses and between RNA processing components that assemble at
various sites on the pre-mRNA are thought to facilitate the efficient production of mRNAs that are suitable substrates for translation. Incorrectly processed transcripts can be recognized as such and degraded (16, 24, 26, 40).
The Drosophila melanogaster suppressor of sable gene,
su(s), encodes a protein involved in nuclear pre-mRNA
metabolism. Loss-of-function su(s) mutations either suppress
or enhance specific mutant alleles of a variety of unlinked genes
(49). Some su(s) mutants also exhibit defects in
viability and male fertility (52). Although both the
su(s) gene and mutant alleles affected by su(s)
have been cloned and characterized, the function of the
su(s) gene product, SU(S), has been somewhat elusive. The
enhanced alleles are associated with large, complex genes that cannot
easily be analyzed in detail at a molecular level. More is known about
the suppressed alleles, through molecular studies of
vermilion (v), yellow (y),
and purple (pr) (20-22, 29). The
su(s)-suppressible mutations have transposon insertions near
the 5' end of the transcribed region that interrupt either the first
exon or the first intron. These mutant genes produce a reduced level of
RNA in su(s)+ flies, and RNA levels are elevated
in su(s) mutants. The RNAs generated initiate at the normal
transcription start site of the genes. Antisense transposon sequences
are incorporated into the pre-mRNA and can be removed during splicing.
In the case of v and y, transposon sequences are
removed inefficiently by splicing at cryptic splice sites near the ends
of the inserted sequences (20, 22). Previous work from this
lab demonstrated that the improvement of a cryptic 5' splice site near
the beginning of a mutant v transgene to a consensus site
increased RNA production from a mutant v transgene, without
improving the splicing efficiency. This change also eliminated the
inhibition of RNA production by SU(S) (21). While these and
other studies have established a connection between SU(S)-mediated
regulation of RNA levels and the efficiency of splicing complex
assembly in the 5' region of the pre-mRNA (21, 29), the
mechanism by which SU(S) regulates accumulation of these RNAs has not
been established. Thus, it is unclear whether SU(S) directly regulates
transcription, splicing complex assembly, or pre-mRNA stability.
SU(S) is a novel, 150-kDa nuclear protein. The initial sequence
analysis (51) defined two regions of SU(S) with similarity to structural motifs found in RNA processing proteins, a highly charged
region in the N-terminal portion of SU(S) and an RNA recognition motif-like motif in the C-terminal region. The importance of these regions to SU(S) function has not been determined. Subsequently, two tandem copies of a CCCH zinc binding domain (57)
of unknown function were identified within SU(S) (43). This
motif is also found in several other eukaryotic proteins, including the
Caenorhabditis elegans transcriptional repressor PIE-1
(6, 43), the mRNA destabilization protein TTP/Nup475/TIS11
(12, 32), the 35-kDa subunit of the splicing factor U2AF
(43, 48), and the 30-kDa subunit of the polyadenylation
factor CPSF (3-5).
Recombinant SU(S), expressed in baculovirus, binds to RNA in vitro;
using a PCR-based binding site selection assay (SELEX), we isolated
high-affinity RNA substrates (45). In this paper, we have
delineated the RNA binding domain of SU(S) and examined its role in
SU(S) function in vivo. Based on the results presented here and
previous studies, we propose that SU(S) binds to the 5' region of the
nascent transcripts via arginine-rich RNA binding motifs (ARMs) and
inhibits RNA production in a way that can be overcome by splicing
complex assembly in the 5' region.
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MATERIALS AND METHODS |
Generation and analysis of MBP-SU(S) fusion derivatives.
In
mapping the RNA binding regions, a 1,084-bp
ClaI-ScaI su(s) cDNA fragment that
encodes the first 360 amino acids of SU(S) was used to generate smaller
fragments by digestion with either appropriate restriction enzymes or
exonuclease III. Subsequently, these fragments were cloned into the
maltose binding protein (MBP) expression vector pPR997 (New England
Biolabs). Deletions of sequences encoding the two ARMs of SU(S) were
introduced using overlapping PCR mutagenesis (25). Unique
XbaI and HindIII restriction sites were
introduced at the site of the ARM1 and ARM2 deletions, respectively, to
facilitate identification of clones containing the desired changes.
Convenient restriction sites were used to introduce fragments containing either one or both deletions into
pMAL- SU(S)1-434, an MBP-SU(S) fusion encoding amino
acids 1 to 434. In addition, small PCR-generated fragments containing
the coding region for ARM1 and ARM2 alone were cloned into pPR997.
Prior to affinity purification of the fusion proteins as described
previously (45), clones were sequenced to ensure that
undesirable alterations were not introduced during the PCRs. RNA
binding activity of the MBP-SU(S) fusions was measured by
nitrocellulose filter binding and Northwestern blot assays as described
previously (45). Kds were determined using SigmaPlot (Jandel Scientific). Under the conditions of these experiments, the Kd is equal to the protein
concentration that results in 50% of the maximal RNA binding.
Construction of clones for germ line transformations.
An
approximately 4-kb XhoI-SpeI cDNA fragment
containing the su(s) coding region from either the wild type
or 
ARM derivatives was cloned into
XhoI-XbaI-digested plasmid pUAST (9).
These clones were injected into yw embryos, and germ line
transformants were isolated essentially as described by Karess
(28). Standard balancer chromosomes were used to establish
homozygous stocks of w+ transformant lines and
to cross the transgenes into the background of su(s) mutants.
Viability studies.
GAL4-expressing stocks were
obtained from the Bloomington Stock Center and crossed to stocks of
transformants carrying UAS-su(s) transgenes. The expression
patterns of the GAL4 drivers are described in Flybase
(http://flybase.bio.indiana.edu). To examine the viability of flies
ectopically expressing SU(S) under control of different GAL4
drivers, crosses were set up with a single vial for each transformant.
The flies were reared at 18°C. All except one of the crosses involved
balanced GAL4 driver stocks, and viability was assessed by
determining the proportion of progeny that lacked the balancer
chromosome. One stock, 1799, was homozygous for the heat-shock
inducible GAL4 driver (hs-GAL4). Thus, all of the
progeny would be expected to express the UAS-su(s)
transgene. Vials yielding normal numbers of progeny were scored as
100% viable; therefore, this value in this particular cross is only an approximation.
The crosses between transformants carrying either a wild-type
su(s) [su(s)wt] or su(s)ARM
transgene and GAL4 stock 2023 were performed in the
following way. Three vials, each containing five pairs of virgin
females and males, were set up for each transformant line tested.
Crosses were performed at 25 and at 18°C; progeny were collected and
scored for 10 and 20 days, respectively.
RNA analysis.
Total RNA was isolated essentially as
described previously (15), and polyadenylated RNA was
purified from the total RNA preparations by using a Poly ATtract mRNA
isolation kit (Promega). Northern analysis was performed as described
previously (21). The probe used for Northern analysis was a
ClaI/DraI su(s) cDNA fragment from
plasmid p15-1, labeled by random priming (2). RNase
protection experiments were performed as described in Current Protocols in Molecular Biology (2). Each reaction
contained 20 µg of total RNA. The probes used were radioactively
labeled, antisense RNAs of an XmaI/BamHI
su(s) cDNA fragment containing portions of exons 3 and 4, and rp49 (21) was used as the internal control.
The probes were labeled by in vitro transcription as described
previously (21).
Protein analysis.
Whole cell extracts were prepared by
grinding 25 to 30 mg of frozen adult flies in 100 µl of 1.5× sodium
dodecyl sulfate (SDS) sample buffer (6× SDS sample buffer is 0.35 M
Tris-Cl [pH 6.8], 10% SDS, 93 mg dithiothreitol per ml, and 30%
glycerol) on ice. Samples were boiled for 10 min prior to a 10-min
centrifugation at 13,000 × g. The supernatant was
assayed for protein content by the Bradford method using the Bio-Rad
protocol. Proteins were resolved by SDS-polyacrylamide gel
electrophoresis, and Western blots were probed with a 1:1,000 dilution
of an affinity-purified polyclonal antibody directed against SU(S)
amino acids 42 to 146. Horseradish peroxidase-conjugated goat
anti-rabbit antibody (Promega) was used as the secondary antibody, and
bands were visualized by enhanced chemiluminescence detection
(Amersham) as recommended by the manufacturer.
Immunocytochemistry.
Polytene chromosome squashes of
third-instar larval salivary glands were prepared and immunostained as
described by Ashburner (1). SU(S) was detected using a 1:400
dilution of affinity-purified polyclonal antibody, directed against
SU(S) amino acids 648 to 808 (45), and a 1:50 dilution of
goat anti-rabbit antibody conjugated with rhodamine as primary and
secondary antibodies, respectively. HRP36 was detected using a 1:600
dilution of mouse monoclonal antibody 5cA5 (a gift from Gideon
Dreyfuss) and a 1:50 dilution of goat anti-mouse secondary antibody
conjugated with fluorescein isothiocyanate. Samples were examined by
confocal microscopy. In double-labeling experiments, individual channel
images were pseudocolored and combined using Photoshop software. For
the whole mounts, salivary glands were fixed in 4.7% formaldehyde for
5 min, washed with phosphate-buffered saline, and immunostained for
SU(S) in essentially the same way as the squashes except that the
incubation with primary antibody was longer (overnight instead of
4 h), and a lower dilution (1:500) of the secondary antibody was
used. The wild-type larvae were from strain Oregon R. The su(s) null mutant stock was su(s)R39 ras
vk. Larvae expressing the su(s) transgenes
were from a su(s)R39 ras vk stock
with a recombinant third chromosome carrying both the
hs-GAL4 driver and a su(s) transgene. The
transgenic stocks also contain the third chromosome balancer TM2; thus,
larvae were either homozygous or heterozygous for the GAL4
driver and the UAS (upstream activation sequence)-su(s) transgene.
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RESULTS |
Two ARMs mediate the specific RNA binding activity of SU(S) in
vitro.
The amino acids responsible for the high-affinity RNA
binding map to the N-terminal 360 amino acids of SU(S) (45).
To define the RNA binding domain more precisely, we expressed cDNA
fragments encoding various portions of this region in Escherichia
coli as MBP fusions (see Materials and Methods). We then measured
RNA binding activity of the affinity-purified fusion proteins using radioactively labeled 473-nucleotide (nt) ftz RNA as the
substrate in either nitrocellulose filter binding or blot overlay
binding assays as described previously (45) (Fig.
1A). These experiments defined two
nonoverlapping RNA binding regions, amino acids 150 to 215 and amino
acids 266 to 309. Examination of the amino acid sequence revealed that
each of these regions contains a high proportion of arginine residues
(Fig. 1A). This suggested that the RNA binding activity of SU(S) might
involve ARMs (11, 56). To determine whether the
arginine-rich regions are required for the RNA binding activity of
SU(S), PCR mutagenesis was used to create precise 25-amino-acid
deletions of sequences encoding residues 151 to 176 (ARM1) and 269 to 294 (ARM2) (Fig. 1B). The deletions were introduced into an
MBP-su(s) cDNA clone encoding the N-terminal 434 amino acids
of SU(S), which also includes the two zinc binding motifs (Fig. 1B).
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FIG. 1.
(A) Delineation of the SU(S) RNA binding domain.
MBP-SU(S) fusion proteins containing different portions of the first
360 amino acids of SU(S) were evaluated for binding to radioactively
labeled ftz pre-mRNA by nitrocellulose filter binding or
blot overlay assays. Plus symbols indicate binding; minus symbols
indicate no binding. The horizontal solid line indicates the region
included in each fusion protein. The shaded vertical bars indicate the
two RNA binding regions defined from this analysis, and the amino acid
sequence of each region is shown beneath the schematic figure. Arginine
residues are indicated in bold type. (B) Amino acid sequence of the
N-terminal 434 amino acids of SU(S). The amino acids deleted in the
SU(S) ARM mutants are shown in bold type. The two zinc binding
motifs are underlined, and the cysteine (C) and histidine (H) residues
that are characteristic of this motif are shown in bold type.
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Nitrocellulose filter binding assays were used to measure the affinity
of the SU(S)
ARM fusion proteins for
ftz RNA and several
small RNAs that were previously isolated by SELEX (
45). The
small RNAs were isolated after eight rounds of SELEX as high-affinity
SU(S) targets from a starting pool of 59-nt RNAs, randomized over
the
central 20 nucleotide positions and with invariant flanking
sequences.
Three of the RNAs tested were from the class of SELEX
RNAs that contain
a close match to the consensus sequence UCAGUAGUCU
(consensus RNAs 8-5, 8-32, and 8-40 [Fig.
2A]). Each consensus
RNA is capable of
forming several possible stem-loops similar
in structure, typically
containing a mismatched base pair in the
stem (Fig.
2A). The SELEX
consensus sequence is predicted to base
pair with sequences near the 5'
end of the RNA (Fig.
2B). The
three other SELEX RNAs tested were also
isolated after eight rounds
of SELEX but lack the consensus sequence
(nonconsensus RNAs 8-10,
8-27, and 8-28 [Fig.
2A]) and are less
capable of forming stem-loops
than the consensus RNAs. The nonspecific
RNA binding activity
of the fusion proteins was measured using a pool
of 59-nt RNAs
that are randomized in the central 20 positions but have
the same
invariant flanking sequences as the SELEX round 8 RNAs.
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FIG. 2.
SELEX RNAs used in the RNA binding assays. (A) Complete
nucleotide sequences of RNAs were isolated as high-affinity substrates
for SU(S) from a pool of 59-nt RNAs with random sequences in the
central 20 positions (round 0) after eight rounds of SELEX
(45). The arrows underneath each sequence define regions of
potential structures as defined by the GCG Stemloop software. The
randomized region of each RNA is defined by underlining. The SELEX
consensus sequence is shown in bold type. The consensus RNAs are 8-5, 8-32, and 8-40; the nonconsensus RNAs are 8-10, 8-27, and 8-28. (B)
Consensus RNA 8-32 with its SELEX consensus sequence paired in
stem-loop structure. The consensus sequence is shown in bold type. (C)
RNA binding activities of the MBP-SU(S) fusion proteins measured in
nitrocellulose filter binding experiments. Kds
shown were determined from the experiments shown in Fig. 3 by using
SigmaPlot and are the averages of two to four independent experiments.
Under the conditions of these experiments, the
Kd is equal to the protein concentration that
results in 50% of the maximal RNA binding. In reactions with very low
levels of binding over the range of protein concentrations tested, a
maximum binding of 60% was assumed in estimating the
Kd.
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Figures
2C and
3 illustrate the results
of RNA binding experiments with these MBP-SU(S) fusion proteins.
Whereas full-length
recombinant SU(S) purified from baculovirus bound
ftz and all
six SELEX round 8 RNAs with similar, high
affinities (apparent
Kd = 2 to 9 nM)
(
45), the binding properties of the MBP-SU(S)
fusion
proteins were more complex. The affinity of the wild-type
fusion
protein [MBP-SU(S)wt] for
ftz and the three consensus RNAs
(apparent
Kd = 4 to 5 nM) was 8- to 22-fold
higher than its affinity
for the nonconsensus RNAs. Likewise, the
fusion protein lacking
ARM2 [MBP-SU(S)
ARM2] bound consensus RNAs
with apparent
Kds of
4.3 to 5.9 nM and
nonconsensus RNAs with 15- to 50-fold-lower
affinity. On the other
hand, the derivative lacking ARM1 [MBP-SU(S)
ARM1]
exhibited a 10- to 20-fold-lower affinity for consensus RNAs (apparent
Kd = 44 to 96 nM) than MBP-SU(S)wt, although it
bound
ftz and
nonconsensus RNAs with
Kds ranging between 12 and 35 nM. The affinity
of each of the MBP-SU(S) proteins for the randomized RNA pool
was
greater than 200 nM, and the derivative that lacks both ARMs
[MBP-SU(S)
ARM1,2] did not bind to any of the RNAs tested.
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FIG. 3.
RNA binding activity of MBP-SU(S) ARM deletion
derivatives. The nitrocellulose filter binding assay was used to
analyze the binding of affinity-purified preparations of MBP-SU(S)
fusion proteins to various radioactively labeled RNAs. SELEX RNAs 8-5, 8-32, and 8-40 are consensus RNAs; SELEX RNAs 8-10, 8-27, and 8-28 are
nonconsensus RNAs. Sequences of the SELEX round 8 RNAs are shown in
Fig. 2. Each set of binding curves shows the activity of a different
MBP-SU(S) fusion protein. (A) MBP-SU(S)wt, which contains the
N-terminal 434 amino acids; (B to D) fusion proteins with deletions of
ARM2, ARM1, and both ARM1 and ARM2, respectively; (E and F) derivatives
with the 25 amino acids of ARM1 and ARM2, respectively, fused to MBP.
Under the conditions of these experiments, the
Kd is equal to the protein concentration that
results in 50% of maximal RNA binding.
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To determine whether either of the arginine-rich regions is sufficient
for RNA binding, we generated MBP fusion proteins that
contained only
the 25-amino-acid arginine-rich segments, ARM1
or ARM2, that were
deleted in the experiments just described.
MBP-ARM1 bound
ftz RNA with an apparent
Kd of
approximately 40
nM and the SELEX RNAs with
Kds
of 200 nM or greater (Fig.
3E).
MBP-ARM2 bound
ftz with a
Kd of about 10 nM and the SELEX RNAs
with
Kds of 60 nM or greater (Fig.
3F). Thus, it
appears that
amino acids outside the regions defined by the ARM
deletions contribute
to the high-affinity binding of SU(S) to the SELEX
RNAs. Furthermore,
these results provide additional confirmation that
the ARM-dependent
RNA binding activity is not due to electrostatic
interactions.
Taken together, these experiments demonstrate that the amino acids
within the regions defined by the ARM deletions mediate
the in vitro
RNA binding activity of SU(S). Whereas either ARM1
or ARM2 is
sufficient for high-affinity binding to the larger
ftz RNA,
the ARM deletions differentially affect binding to the
smaller SELEX
RNAs. ARM1 is required for high-affinity binding
to consensus RNAs, and
ARM2 mediates binding to the nonconsensus
RNAs. Since both the SU(S)
zinc binding motifs are intact in the
MBP-SU(S)
ARM1,2 fusion
protein, which does not bind RNA, these
motifs, by themselves, do not
mediate RNA binding in
vitro.
Overexpression of SU(S) can be lethal in vivo.
Having
established that the ARM deletions eliminate the in vitro RNA binding
activity of SU(S), we wanted to assess the consequences of these
alterations on SU(S) function in vivo. For this analysis, a wild-type
full-length su(s) cDNA and derivatives with the ARM deletions described above were constructed and introduced into flies by
germ line transformation. A suitable su(s) promoter fragment capable of driving expression of a su(s) cDNA had not
been defined. Therefore, we ligated the su(s) coding
region to a promoter fragment containing UASs that are responsive to
the transcriptional activator GAL4 (9). Transformants were
isolated under conditions where the UAS-su(s) transgenes
were transcriptionally inactive. Subsequently, ectopic expression of
the transgenes was activated by performing a genetic cross to introduce
a GAL4 transgene (GAL4 driver).
Because initial experiments indicated that ubiquitous expression of a
wild-type
su(s) transgene was lethal (data not shown),
we
examined the viability of flies expressing the
UAS-su(s)wt transgene under the control of six different
GAL4 drivers
(Table
1). This analysis was performed in
the background of wild-type
endogenous
su(s). Analysis of
the progeny recovered from these
crosses demonstrated that different
ectopic SU(S) expression patterns
are lethal to variable degrees (Table
1). No progeny that carried
both
UAS-su(s)wt and the
ubiquitously expressed
e22c-GAL4 driver
were recovered. The
viability of
UAS-su(s)wt transformants containing
T80-GAL4, which is ubiquitously expressed in imaginal discs,
varied
widely, with no
T80-GAL4/UAS-su(s)wt progeny being
recovered in
more than half of the transformant lines tested (data not
shown).
The variation in the severity of the viability defect most
likely
reflects position-dependent differences in the expression levels
of the
UAS-su(s)wt transgene, presumably with higher levels
of
expression producing more pronounced effects. In contrast to the
results obtained with ubiquitously expressed
GAL4 drivers,
flies
carrying
hs-GAL4 and
UAS-su(s) were
recovered at the usual frequency
when reared in the absence of heat
shock. The other
GAL4 drivers
(
sev-GAL4,
69B-GAL4, and
30A-GAL4), which generate
restricted
GAL4 expression patterns, produced relatively
modest effects on
viability. Thus, this analysis clearly indicates that
overexpression
of SU(S) is lethal. Analysis of survival at different
developmental
stages did not reveal a clear-cut lethal period; however,
all
of the developing flies died prior to pupation (data not shown).
We also examined the viability of flies expressing
UAS-su(s)ARM single- and double-deletion mutant
transgenes. As was seen
with the wild-type
su(s) transgene,
no
e22c-GAL4/UAS-su(s)ARM progeny were recovered with any
of the derivatives tested (data
not shown). Thus, the lethality caused
by ubiquitous, high expression
depends on a region of SU(S) other than,
or in addition to, the
ARMs. The viability of flies carrying the
wild-type and ARM deletion
mutants was also examined in the background
of the
sev-GAL4 driver.
Expression of the
sev-GAL4 transgene is controlled by a hybrid
promoter,
consisting of the
hsp70 TATA box and the
sevenless enhancer.
This promoter directs
GAL4
expression primarily in eye discs (
47).
Examination of
expression of a
UAS-lacZ reporter gene under control
of
sev-GAL4 showed that this driver directs a low level of
expression
of a
UAS promoter in other larval tissues as well
(our unpublished
observations). Because the viability defects produced
by this
driver are relatively subtle, multiple transformant lines were
analyzed for each
UAS-su(s) transgene. Furthermore, to
distinguish
defects related to
UAS-su(s) expression from
those caused by disruption
of a gene at the site of the transgene
insertion, parallel experiments
were performed both at 25 and 18°C.
Because GAL4 is less active
at lower temperatures, viability defects
related to the level
of ectopically expressed SU(S) are expected to be
more severe
at 25 than 18°C (
9), whereas defects related
to gene disruption
at the site of the transgene insertion are unlikely
to be affected
by
temperature.
In these experiments, differences were observed in the viability of
flies expressing the
su(s)wt and various
su(s)ARM transgenes
(Table
2). Flies expressing
su(s)wt
were recovered at 19 to 59%
of the expected frequency at 25°C and at
higher frequencies at
18°C. Thus, consistent with the analysis
described earlier, expression
of the wild-type transgene reduces
viability. On the other hand,
two of the three transformant lines
expressing
su(s)ARM1 and
all three lines expressing
su(s)ARM1,2 were recovered at close
to the expected
frequencies at both temperatures, indicating that
these transgenes do
not negatively affect viability. The viability
defect observed in one
UAS-su(s)ARM1 transformant line (28A [Table
2])
appeared to be related to disruption of a gene at the site
of the
UAS-su(s) transgene insertion because flies expressing
the
transgene were recovered at similar, low frequencies at both
temperatures. Two of the three lines expressing
su(s)ARM2
were
recovered at frequencies that were not significantly different
from the wild-type level at 25°C. However, all three
su(s)ARM2-expressing
lines were recovered at a lower than
expected frequency at 18°C.
Taken together, these results indicate
that the ARMs mediate one,
but not the only, component of the lethal
effect of overexpressing
SU(S). Furthermore, the lower viability of
su(s)ARM2-expressing
flies at 18°C, the temperature at
which less protein should be
produced, suggests that deletion of ARM2
increases the detrimental
activity of SU(S) at 18°C.
Examination of the eye color of flies carrying the hypomorphic
su(s)51c15 allele, the suppressible
vk allele, the
UAS-su(s)wt cDNA
transgene, and the
hs-GAL4 driver
revealed that the
UAS-su(s)wt cDNA transgene did not provide
su(s) function sufficient to rescue the
v eye color phenotype at
25°C
(data not shown). At higher temperatures, heat shock-induced
expression
of
UAS-su(s)wt was lethal. Other
GAL4-driven
su(s) expression
patterns either did
not rescue or gave ambiguous results. Thus,
this expression system was
unsuitable for determining whether
the ARMs are required for the
function of SU(S) in regulating
expression of mutant
v
alleles. These observations, together with
the ectopic expression
induced lethality, indicate that the normal
regulatory activity of
SU(S) is highly dependent on the level
and pattern of
su(s) expression.
ARM1 is required for the polytene chromosome association of
SU(S).
Endogenous SU(S) is found both in the extrachromosomal
compartment of the nucleus and at discrete sites on larval salivary gland polytene chromosomes (45). When polytene chromosomes
from wild-type flies are stained by indirect immunofluorescence with anti-SU(S) polyclonal antibodies, a strong signal is observed at fewer
than 20 chromosomal sites whereas many other sites give a weak signal.
Since SU(S) is an RNA binding protein, the chromosomal localization is
likely to represent SU(S) binding to nascent RNA transcripts, although
it is possible that SU(S) also interacts with one or more
chromatin-associated components. To test whether the ARM deletions
affect the chromosomal association of SU(S), we used immunofluorescence
to examine polytene chromosomes from larvae expressing wild-type and
ARM deletion UAS-su(s) transgene derivatives under control
of the hs-GAL4 driver, which is expressed in salivary glands
even in the absence of heat shock (Fig.
4). This analysis was performed in the
background of an endogenous su(s)R39 null
mutant, which gives no immunofluorescence signal with the anti-SU(S)
antibodies used (Fig. 4B). Polytene chromosomes were simultaneously
labeled with polyclonal anti-SU(S) polyclonal antibodies and with a
monoclonal antibody (5cA5) that recognizes HRP36, one of the abundant
hnRNP proteins in Drosophila. In this experiment, anti-HRP36
was used solely as a positive control for the immunostaining procedure.
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FIG. 4.
Immunolocalization of SU(S) ARM mutant derivatives on
third-instar larval salivary gland polytene chromosomes. Shown are
confocal images of chromosome squashes double labeled by indirect
immunofluorescence with antibodies that recognize SU(S) and the
positive control HRP36. The pseudocolored images indicate SU(S) in red
(A to F) and HRP36 in green (A' to F'). HRP36 is found at a much larger
number of bands than endogenous SU(S), and there is little overlap
between strong HRP36 sites and strong SU(S) sites. (A and A')
su(s)+; (B and B')
su(s)R39 null mutant. The remaining images were
prepared from larvae grown at room temperature, expressing an SU(S)
cDNA transgene under control of the hs-GAL4 driver in
su(s) null mutant background: (C and C') SU(S)wt; (D and D')
SU(S)ARM1; (E and E') SU(S)ARM2; (F and F') SU(S)ARM1,2.
|
|
A larger number of chromosomal sites stained strongly with the
anti-SU(S) antibodies in chromosome squashes prepared from
larvae
expressing SU(S)wt (Fig.
4C) and SU(S)
ARM2 (Fig.
4E) than
in larvae
lacking the transgene (Fig.
4A). The stronger signal
at least in part
reflects the higher level of SU(S) produced by
the transgenes (see
below). In contrast, no signal was observed
when chromosomes expressing
SU(S)
ARM1 were stained with anti-SU(S)
antibodies (Fig.
4D). Results
obtained with chromosomes from transformants
expressing the
double-deletion derivative SU(S)
ARM1,2 were somewhat
variable. In
some experiments no chromosomal association of SU(S)
ARM1,2
was
detected (data not shown), whereas in other experiments weak
staining
was observed. As described below, SU(S)
ARM1,2 is expressed
at very
high levels in comparison to the other SU(S) derivatives.
Thus, it is
possible that SU(S) interacts weakly with polytene
chromosomes
independent of its RNA binding
motifs.
One possible explanation for the failure of SU(S)
ARM1 and
SU(S)
ARM1,2 to associate with polytene chromosomes might be that
the
ARM1 deletion blocks entry of these proteins into the nucleus.
Immunofluorescence labeling of whole salivary gland cells demonstrated
that each of the SU(S) derivatives accumulates in the nucleus
as
expected (Fig.
5). Another possibility is
that the proteins
are unstable. However, Western blot analysis (data
not shown;
see below) indicates that the proteins produced by the
transgenes
accumulate at higher levels than endogenous SU(S). Based on
these
results, we conclude that ARM1, which promotes binding to SELEX
consensus RNAs in vitro, is required for the stable association
of
SU(S) with polytene chromosomes in vivo and that ARM2 alone
is neither
sufficient nor required for this interaction.
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FIG. 5.
Nuclear localization of SU(S)ARM derivatives in salivary
gland cells. Shown are confocal images of whole mounts of third-instar
larval salivary glands labeled by indirect immunofluorescence with a
polyclonal anti-SU(S) antibody. Like endogenous SU(S), the four SU(S)
derivatives localize in the nucleus. (A) su(s)+;
(B) su(s) null mutant. (C to F) Images prepared from larvae
expressing various SU(S) transgenes under control of the
hs-GAL4 driver in su(s) null mutant background:
(C) SU(S)wt; (D) SU(S)ARM1; (E) SU(S)ARM2; (F) SU(S)ARM1,2.
|
|
SU(S)wt represses RNA accumulation from the su(s) cDNA
transgenes.
We used Western blots to compare the levels of SU(S)
produced by the different transgenes. Quite unexpectedly, we found that SU(S)ARM1,2 accumulates at a much higher level than SU(S)wt (Fig. 6A). This higher level of mutant protein
accumulation was observed with more than 10 different transformant
lines (data not shown) and with three different GAL4 drivers
(data not shown). Thus, the difference is not likely to be related to
position-dependent variation in transcription of the transgene or
tissue-specific effects. The accumulation level of SU(S) derivatives
with single ARM deletions varied between transformant lines. In some
transformants, SU(S)ARM1 or SU(S)ARM2 accumulated at similar
levels as SU(S)wt. In other transformants, the single deletion
derivatives accumulated at levels intermediate levels, i.e., higher
than SU(S)wt but substantially lower than SU(S)ARM1,2 (data not
shown).
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FIG. 6.
Levels of su(s)wt and
su(s)ARM1,2 expression. (A) Western blot of total protein
(200 µg per lane) extracted from balanced stocks with a recombinant
third chromosome carrying both a UAS-su(s) transgene and the
hs-GAL4 driver. The blot was probed with an anti-SU(S)
polyclonal antibody. (B) Northern blot of polyadenylated adult RNA (2 µg per lane) isolated from the same flies as in panel A probed with
su(s) and rp49 cloned sequences.
|
|
We performed Northern blot analysis on poly(A)
+ mRNA
isolated from adult flies carrying the wild-type and double-mutant
su(s) transgenes in the background of the
su(s)R39 allele (Fig.
6B). This analysis showed
that the difference also
occurs at the RNA level; i.e.,
su(s)ARM1,2 transcript accumulates
at a higher level than
su(s)wt mRNA transcript. We reasoned that
the difference in
RNA levels could be due to either nucleotide
sequence differences
between the wild-type and mutant RNAs or
a difference in the ability of
the wild-type and mutant SU(S)
proteins to regulate the amount of RNA
generated from the transgenes.
To distinguish between these
possibilities, we designed a
cis-trans test and performed a
cross to introduce both transgenes into the
same cells. If the mutant
RNA accumulates at a higher level because
it is inherently more stable
(a
cis effect), then the difference
in RNA levels will also
occur when both transgenes are expressed
in the same cells. On the
other hand, if SU(S) regulates accumulation
of these RNAs (a
trans effect), then the
su(s)wt and
su(s)ARM1,2 RNAs will be found at similar
levels in cells that express both
proteins. In the latter case, the
level of the two RNAs would
depend on the degree to which
SU(S)
ARM1,2 interferes with the
activity of
SU(S)wt.
We performed RNase protection analysis to compare the levels of the
su(s)wt and
su(s)ARM1,2 RNAs in flies carrying
one copy
of either the wt or mutant transgene and flies carrying one
copy
of both transgenes (Fig.
7). This
analysis was performed in the
background of the
su(s)R39 null mutant. To distinguish between the
su(s)wt and
su(s)ARM1,2 transcripts, we used a
wild-type, antisense
su(s) RNA probe that
spans the ARM
coding region. After hybridization and RNase treatment,
su(s)wt and
su(s)ARM1,2 transcripts generate
394- and 262-nt
protected fragments, respectively. As shown in Fig.
7,
the level
of
su(s) transcript in RNA prepared from flies
carrying only the
mutant transgene was fivefold higher than the level
observed in
flies that were carrying only the
su(s)wt
transgene. In contrast,
in RNA prepared from flies expressing both the
wild-type and
ARM1,2
transgenes, both
su(s) RNA types
accumulated at a similar level.
This demonstrates that SU(S) protein
regulates accumulation of
RNA from the
UAS-su(s) cDNA
transgenes and that the ARMs mediate
this regulation. In flies
expressing both transgenes, the level
of
su(s)wt RNA was
1.5-fold higher than the level of this RNA
in flies carrying only the
wild-type transgene. The level of
su(s)ARM1,2 RNA was
twofold lower in flies carrying both transgenes than in
flies
expressing only the
su(s)ARM1,2 transgene. These
intermediate
levels suggest that both the wild-type and ARM deletion
derivatives
of SU(S) can influence RNA production from the transgenes
and
that there is not a clear dominance relationship between the
wild-type
and mutant forms of the protein.
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|
FIG. 7.
RNase protection analysis of su(s)wt and
su(s)ARM1,2 RNA levels. Total RNA was isolated from adult
flies reared at 25°C. Flies with one copy of a transgene contained
the third chromosome balancer TM2 and a recombinant third chromosome
carrying a UAS-su(s) transgene and the hs-GAL4
driver. Flies with both transgenes contained a wild-type transgene on
one of the third chromosomes and a mutant transgene on the other. The
transgenic flies also carry the null su(s)R39
allele on the X chromosome and thus produce no endogenous
su(s) protein or mRNA; 20 µg of total RNA was used in each
reaction. The 447-nt hybridization probe in this experiment was
prepared from a wild-type su(s) cDNA clone and spans the
region that encodes the ARMs (see Materials and Methods). The
su(s)wt and su(s)ARM1,2 protected fragments
are 394 and 262 nt, respectively. Indicated beneath the lanes are the
relative RNA levels. The values shown are the average of three separate
experiments performed with the same RNA preparations. The standard
deviations were less than 10% of the values shown.
|
|
In attempt to test whether
su(s)ARM1,2 is capable of
elevating the amount of RNA produced from endogenous
su(s),
we crossed
the
GAL4-driven
UAS-su(s)ARM1,2
transgene into the background
of a wild-type endogenous
su(s) allele and performed Northern
analysis with a probe
that could detect only the endogenous
su(s) transcript. We
observed no effect of the
su(s)ARM1,2 transgene
on the
level of endogenous
su(s) RNA (data not shown). This
could
indicate that SU(S)
ARM1,2 is not capable of
affecting regulation
of the endogenous
su(s)+
gene. However, the absence of an effect might be because SU(S)
ARM1,2
produced by the
GAL4-driven transgene is not expressed in
the
appropriate tissue or at the level needed to effect expression
of
endogenous
su(s). Thus, no definitive conclusion could be
drawn
from this
experiment.
| |
DISCUSSION |
We have shown that two ARMs mediate the in vitro RNA binding
activity and in vivo function of SU(S). ARM1 is required for high-affinity binding of SU(S) to SELEX consensus RNAs, whereas ARM2
promotes binding at roughly a 10-fold-lower affinity to SELEX nonconsensus RNAs. The CCCH zinc binding motifs, located just downstream of the ARMs, are incapable of promoting stable RNA binding
in vitro, although they may influence the stability of the SU(S)-RNA
interaction in vivo. The SELEX consensus RNAs contains a close match to
the sequence UCAGUAGUCU, flanked by GU-rich sequences. Previous in vitro RNA footprinting experiments showed that recombinant, baculovirus-expressed SU(S) interacts with nucleotides of the consensus
and GU-rich regions (45). The SELEX consensus sequence is
complementary to invariant sequences near the 5' end of the SELEX RNA.
Thus, one explanation for the repeated isolation of RNAs containing
this sequence is that it enables the RNA to fold into a hairpin
structure recognized by SU(S), rather than representing a particular
sequence that is bound by SU(S). However, the sequence composition of
the stem-loop might be a factor in SU(S) binding. For example, as
previously noted (45), the SELEX consensus sequence resembles the 5' splice site consensus MAGGURAGU,
where M denotes C or A, R denotes A or G, and the underlined GU is the
invariant dinucleotide found at the 5' boundary of the intron
(44) and the sequence commonly found near the transcription
initiation site (UCAGU) (14). Perhaps SU(S) binds to
stem-loops in regions of a pre-mRNA that includes these sequences,
i.e., near the cap site and/or 5' splice site. Additional work will be
required to determine the relative importance of RNA sequence versus
structure in the binding activity mediated by ARM1.
The ARMs of SU(S), particularly ARM1, exhibit the usual features that
are characteristic of this class of RNA binding motif. ARM-RNA binding
domains are typically short, i.e., 10- to 20-amino-acid, arginine-rich
regions that recognize particular structural features of RNA rather
than a specific sequence (11). The most extensively studied
ARM proteins, including N protein of bacteriophage lambda, Nun protein
of bacteriophage HK022, and human immunodeficiency virus Tat and Rev,
bind near the 5' end of their target transcripts (34, 39, 50, 54,
55) and regulate either transcription elongation or RNA export
(17-19).
Previous work has shown that several su(s)-suppressible
alleles have antisense transposon insertions in the 5' region of their genes that disrupt either the first exon or the first intron
(20-22, 29). A higher level of RNA is produced by these
mutant alleles in su(s) mutants than in
su(s)+ flies. In our earlier studies, we
proposed that the regulation of RNA accumulation by SU(S) was at the
level of RNA stability, because the effect was related to splicing
complex assembly, which at the time was thought to occur
posttranscriptionally. However, since transcription and RNA
processing are coupled, it is possible that SU(S) binding to RNA
affects transcription. For example, perhaps SU(S) binding
stabilizes stem-loop structure in the 5' region of the pre-mRNA, which,
in turn, pauses the elongating transcription complex. Splicing complex
assembly in the 5' region of the pre-mRNA might release the paused RNA
polymerase II just as ribosome binding to a nascent transcript releases
the paused prokaryotic RNA polymerase to synchronize transcription and
translation (13). Suppressible mutant alleles contain
transposon insertions near their 5' regions and thus lack normal
splicing signals near the transcription start sites. Expression of
these insertion mutant alleles would, thus, be irreversibly repressed
by SU(S) binding. This type of role for SU(S) in regulating
cotranscriptional splicing complex assembly is analogous to the
proposed role of SPT5 and its homologues in repressing transcription
elongation to couple capping to transcription (23, 53).
Studies currently under way are designed to test whether SU(S)
regulates transcription or RNA stability.
In this report, we have demonstrated that SU(S) negatively regulates
production of transcripts from the UAS-su(s) cDNA
transgenes. Thus, we have identified the first example of regulation by
SU(S) that does not involve mutant transcripts with a transposon
insertion. This effect cannot be explained in terms of our previous
model that SU(S) binds cryptic splice sites to promote recognition of authentic splice sites because the only intron present in these constructions is a small simian virus 40 intron, which was introduced into the 3' untranslated region during subcloning of the cDNA into the
transformation vector (9). Like the mutant v and
y alleles that are negatively regulated by SU(S), the first
intron of the su(s) cDNA transgenes is a long distance, 5 kb, downstream of the transcription start site. In most
intron-containing pre-mRNAs, the first intron is located near the
beginning of the transcribed region (41), and an intron in
this position appears to be important for expression at a high level.
Studies in a variety of different systems indicate that cDNA transgenes
are expressed at lower levels than their genomic counterparts in vivo
and that expression of a cDNA can be elevated by inclusion of an intron
near the 5' end of the gene (10, 30, 31, 37, 38, 46). The
magnitude of this effect varies, depending on sequences present in the
RNA and the promoter that is used to direct transgene expression
(33, 35). Furthermore, in a study that examined the factors
that influence intron-dependent enhancement of gene expression, the strength of the 5' splice site was found to be important
(31). Likewise, we previously showed that improvement of a
cryptic 5' splice site near the beginning of a mutant v
transgene to a consensus site increased RNA production from a mutant
v transgene, without improving the splicing efficiency. In
addition, SU(S) does not inhibit the production of RNA from the
transgene with a consensus 5' splice site near the beginning of the
transcribed region but does limit production of RNA from a similar
transgene that lacks a strong 5' splice site in this region
(21). In light of these observations, it seems plausible
that SU(S) is a component of the intron-dependent gene expression
pathway and that studies of SU(S) function will provide insights into
the general mechanism that limits expression of cDNA transgenes and
antisense RNAs in vivo. The su(s) gene is nonessential,
although su(s) mutations impair viability and fertility to
variable degrees (36). However, our finding that
overexpression of SU(S) can be lethal suggests that SU(S) functions in
an essential process and that the level of SU(S) in the nucleus is an
important aspect of its regulatory activity.
| |
ACKNOWLEDGMENTS |
We thank G. Dreyfuss for providing monoclonal antibody 5cA5. We
thank G. Maroni and M. Peifer for critical comments on the manuscript
and R. Kole for a helpful discussion. We are grateful to S. Whitfield
for assistance with the figures.
This work was supported by grants MCB-950631 and MCB-9808150 from the
National Science Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, CB# 3280, UNC at Chapel Hill, Chapel Hill, NC 27599-3280. Phone: (919) 966-4989. Fax: (919) 962-1625. E-mail:
lsearles{at}emailunc.edu.
Present address: Department of Botany, North Carolina State
University, Raleigh, NC 27695.
| |
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Molecular and Cellular Biology, November 2000, p. 8198-8208, Vol. 20, No. 21
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Abstract
Introduction
