Department of Biology and Center for Complex Systems, MS 008, Brandeis
University, Waltham Massachusetts 02454, USA
* Present address: Department of Anatomy and Neurobiology, Washington University
School of Medicine, St Louis, MO 63110, USA
Author for correspondence (e-mail:
white{at}brandeis.edu
)
Accepted 3 April 2002
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Summary |
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Key words: ARE, Transgenic studies, Neuron-specific, Alternative splicing, Transcript stability, RRM
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Introduction |
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The predominant function so far associated with the ELAV/Hu-family proteins
is to act as regulators of mRNA stability through their interaction with the
previously defined AU-rich elements (AREs) that usually reside within the
3' untranslated region (UTR) of the transcripts. ARE sequences were
identified as being important in the rapid degradation of many mRNAs including
the transcripts of the immediate early response gene c-fos and human
granulocyte macrophage colony stimulating factor gene
(Chen and Shyu, 1995). AREs are
AU rich, generally but not always contain multiple copies of AUUUA
pentanucleotide, and range in size between 50-150 nucleotides
(Chen and Shyu, 1995
). Current
evidence indicates that the neuronal vertebrate ELAV-proteins, Hel-N1, HuC and
HuD, as well as the ubiquitously expressed HuR stabilize ARE-containing mRNAs
(Levine et al., 1993
;
Brennan and Steitz, 2001
;
Keene, 1999
). Interestingly,
RBP9, a Drosophila protein, is reported to destabilize transcripts
through ARE sequences (Kim-Ha et al.,
1999
; Park et al.,
1998
).
ELAV/Hu-family proteins are composed of three classic RRM (RNA recognition
motif) domains, and a hinge region separates the first two tandem RRMs from
the third RRM. Recent studies have shown the hinge region to include nuclear
import/export signals (Atasoy et al.,
1998; Fan and Steitz,
1998
; Yannoni and White,
1999
). The hinge region is also likely to be important for
protein-protein interactions and may conceivably provide some of the
functional specificity to individual members of this family.
ELAV, a Drosophila neural-specific protein, provides a vital
function that is essential to the development and maintenance of neurons.
Studies in our laboratory have demonstrated that ELAV is essential to the
formation of neural specific splice forms of three genes, neuroglian
(nrg), erect wing (ewg) and armadillo (arm), in
Drosophila (Koushika et al.,
2000). These three genes regulated by ELAV are ubiquitously
expressed, but also generate neural-specific isoforms. The two isoforms of
nrg, neural-specific Nrg180 and ubiquitous
Nrg167, are generated by 3' exon choice. In the default
splicing, the common exon is spliced to the penultimate exon, while in
neurons, at least some splicing occurs to the last exon yielding a
neural-specific isoform. We have shown that the regulated alternatively
spliced intron of nrg (nASI), which spans the sequence
between the common and the neural-specific exon, contains all the sequences
necessary for the splicing regulation
(Lisbin et al., 2001
).
Despite their extensive homology at the amino acid level, based on current
data, Drosophila ELAV and human HuD
(Szabo et al., 1991) have been
associated with different functions. The amino acid identity between these
proteins for RRM1, RRM2 and RRM3 is 74%, 63% and 71%, respectively. The main
difference appears to be in the hinge region where only 16% identity is seen.
In addition to the different functions that these proteins serve, they show
distinct patterns of localization within cells. The neuron-specific HuD is
primarily cytoplasmic in cultured cell lines, but it appears to be equally
distributed in both the cytoplasm and nucleus in neurons of the hippocampus
(reviewed by Antic and Keene,
1997
). The transgenic expression of HuD in Drosophila
neurons also shows cytoplasmic expression; however, endogenous or
transgene-expressed ELAV is mainly nuclear
(Yannoni and White, 1999
).
Also, ectopically expressed ELAV in Xenopus neural tube cells
localizes primarily to the nuclei (Perron
et al., 1997
).
Intrigued with the functional differences between ELAV and HuD, we
investigated and compared their properties in the same cells on the same RNA
substrates in wing imaginal disc cells of Drosophila. We chose wing
imaginal disc cells because they are devoid of endogenous ELAV, and because
our previous studies show that ectopic ELAV expression in these cells is
sufficient for the generation of the neural-specific isoforms of nrg,
ewg and arm, the three known ELAV-regulated genes
(Koushika et al., 2000). ELAV
and HuD were ectopically expressed in the wing discus using the GAL4/UAS
system as described (Brand and Perrimon,
1993
; Koushika et al.,
1996
). Effects of ELAV and HuD expression were assayed in
transgenic fly strains carrying transgenes in which generally expressed
Ubiquitin-63E (Ubi-p63E) promoter
(Lee et al., 1988
) was used to
drive green fluorescent protein (GFP) coding sequence. Transgenes with introns
in the ORF were constructed with either nASI, a regulated
alternatively-spliced intron from the nrg gene, or GI, a
constitutively-spliced artificial intron
(Mottes and Iverson, 1995
). In
the nASI- or GI-containing transgenes, the splicing of the intron was
essential for GFP expression. To study the effect of 3'UTR, each
transgene was constructed with two distinct 3'UTRs from
Drosophila genes, Heat-shock-protein-70Ab (Hsp70Ab) and
Actin 5C (Act5C). Our objective was to examine (1) whether HuD, like
ELAV, is capable of inducing neural-specific splicing of the nASI in
non-neural tissue or the two differ in this ability; and (2) if
ectopically-expressed HuD and/or ELAV influence the level of GFP produced from
the reporter genes through distinct 3'UTRs.
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Materials and Methods |
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To create pP{UgGA} and pP{UgGH}, the GI intron was PCR
amplified from pP{GI} (Mottes and
Iverson, 1995) using the primers
5'-GCGGTACCCAGGTAAGTTAGTAGATAG-3' and
5'-AACGACGGGATCGTTTGC-3'. The GI intron fragment was
KpnI/NotI-digested and inserted into the KpnI and
NotI sites of pP{UGA} to generate pP{UgGA}. The
same GI intron fragment was inserted into the KpnI and NotI
sites of pP{UGH} to create pP{UgGH}. In both
pP{UgGA} and pP{UgGH}, the GI intron is inserted between the
Myc-tag and GFP coding sequences so that the GFP expression is
dependent on GI intron splicing.
To construct pP{UnGA} and pP{UnGH}, an
oligonucleotide-mediated site-directed mutagenesis
(Kunkel et al., 1987) was
performed on an nASI cloned in pBluescript (Stratagene). The
oligonucleotide 5'-AATCCGTGAGTTCAGGTACCAAACCGGGCGTGG-3' was used
for the mutagenesis to create a KpnI site upstream of the
nASI. The nASI was excised from the vector by cutting with
KpnI and NotI, and cloned between the KpnI and
NotI sites of pP{UGA} generating pP{UnGA}.
Similarly, the KpnI-NotI nASI fragment was inserted
into the KpnI and NotI sites of pP{UGH} to give
pP{UnGH}. Both pP{UnGA} and pP{UnGH} have the
nASI between the Myc-tag and GFP coding sequences. In both
cases, the expression of GFP requires neural splicing of nASI.
Germline transformation
Df(1)w/y w; Ki pp2-3/+ embryos were injected with
the construct DNA (Robertson et al.,
1988
; Rubin and Spradling,
1982
). The germline transformants were recovered based on
[w+] eye color, and transgenic lines were established by
standard procedures.
Genetic crosses
Flies were reared at 25°C. For nomenclature of transgenes, see
Table 1; for structure of
transgenes see Fig. 1.
|
|
To examine the effect of HuD and ELAV ectopic expression on GFP reporter expression, UxGX/CyO; dpp-GAL4/TM6B Tb females were crossed to UAS-elav2e2 or UAS-HuD7a; UAS-HuD6c males, where UxGX represents one of the following GFP reporters: UGA, UGH, UgGA, UgGH, UnGA and UnGH. UAS-HuD7a and UAS-HuD6c are different insertions of the same transgene. Progeny larvae, ectopically ELAV-expressing (genotype UxGX/UAS-elav2e2; dpp-GAL4/+) or ectopically HuD-expressing (genotype UxGX/UAS-HuD7a; dpp-GAL4/UAS-HuD6c) were identified by GFP expression and Tb+ phenotype, and used for GFP fluorescent analysis and in situ hybridization. UxGX/+ larvae were used as controls, and UAS-GFP/+; dpp-GAL4/+ larvae were used to show the dpp-GAL4 expression pattern.
For imunofluorescent ectopic ELAV and HuD localization, dpp-GAL4/TM6B Tb females were crossed to UAS-elav2e2 or UAS-HuD7a; UAS-HuD6c males to obtain UAS-elav2e2/+; dpp-GAL4/+ (ectopic ELAV expressing) and UAS-HuD7a/+; dpp-GAL4/UAS-HuD6c (ectopic HuD expressing) larvae which were identified by Tb+ phenotype.
Fluorescence analysis
For larval fillet preparations, wandering third instar larvae were
dissected in a dissecting chamber containing phosphate-buffered saline (PBS)
and the gut and Malpighian tubules were removed. The dissected larvae were
washed in PBS, fixed in 4% formaldehyde in PBS for 5 minutes at room
temperature (RT), washed in PBS, then mounted with 70% glycerol in PBS.
Photographs were taken using Leica MZFLIII dissection microscope and Nikon
N6006 camera loaded with Kodak Elite Chrome 400 film.
To analyze GFP expression, wing discs of wandering third instar larvae were dissected in PBS, fixed in 4% formaldehyde in PBS for 5 minutes at RT, washed in PBS, then mounted with 70% glycerol in PBS. GFP fluorescence was analyzed under Zeiss Axiophot fluorescence microscope. Fluorescence images were obtained using Roper Scientific SenSys CCD camera. At least 20 discs of each genotype were analyzed.
For immunofluorescent analysis, wing discs of wandering third instar larvae were dissected in PBS and fixed in 4% formaldehyde in PBS for 1 hour at RT. The wing discs were washed in 0.3% Triton X-100 in PBS (PBTx), treated with 100 µg/ml RNase A in PBTx for 30 minutes at 37°C, washed in PBTx, and blocked in 5% normal goat serum for 30 minutes at RT. Mouse anti-ELAV mAb 7D and mouse anti-HuD mAb 16A11 (a gift of H. Furneaux) were used as primary antibodies at dilutions of 1:100 and 1:250, respectively, in PBTx. A fluorescein-conjugated goat anti-mouse immunoglobulin antibody (Jackson ImmunoResearch Laboratories) was used as secondary antibody at a dilution of 1:100 in PBTx. Primary and secondary antibody incubations were carried out overnight at 4°C. Nuclear staining was achieved by mounting the tissues with 70% glycerol in PBS containing 1 µg/ml propidium iodide. Confocal images were collected on a Bio-Rad MRC-600 confocal microscope.
In situ hybridization
A digoxigenin-labeled antisense RNA probe was prepared by in vitro
transcription using digoxigenin-11-UTP (Boehringer Mannheim) and T3 RNA
polymerase (Boehringer Mannheim). A GFP cDNA cloned into
pBluescript plasmid (Stratagene) was used as a template. The probe
size was decreased as described (Lehmann
and Tautz, 1994).
Anterior halves of wandering third instar larvae were dissected and
inverted in PBS, then fixed in 4% formaldehyde in PBS for 25 min at RT. The
fixed larval heads were washed in PBS containing 0.1% Tween 20 (PBTw), treated
with 8 µg/ml proteinase K in PBTw for 10 min at RT, washed in PBTw, and
fixed in 4% formaldehyde in PBTw for 25 min at RT. Hybridization of the
GFP RNA probe was performed as described
(Lehmann and Tautz, 1994).
Hybridization signals were detected with an anti-digoxigenin Fab fragments
conjugated to alkaline phosphatase (Boehringer Mannheim) using 4-nitroblue
tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer
Mannheim) as substrates. The stained wing discs were dissected and mounted
with 70% glycerol in PBS. Photographs were taken using Zeiss Axioskop
microscope and Zeiss MC80 camera loaded with Kodak Elite Chrome 160T film.
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Results |
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Reporter genes UGA, UnGA and UgGA have Act5C
3'UTR, and UGH, UnGH and UgGH have Hsp70Ab
3'UTR. We chose Hsp70Ab trailer because it potentially has an
ARE-like sequence structure. It is AU-rich (76% AU) and it has four AUUUA
motifs (Fig. 2A). Moreover, it
is known that the transcripts of Drosophila Hsp70 genes are highly
unstable at normal temperature, and at least for one of the Hsp70
genes, the signal for rapid degradation resides in its 3'UTR
(Petersen and Lindquist,
1989). The Act5C trailer was chosen for comparison
because it does not have ARE-like characteristics; it has a lower AU content
(58%) and has a single AUUUA motif (Fig.
2B). Although no direct analysis on stability of Act5C
trailer has been published, Act5C mRNA is likely to be stable
compared with Hsp70 mRNAs. In Drosophila oocytes
Act5C transcripts are more stable than Heat-shock-protin-83,
nanos and string transcripts, three transcripts that are known
to be highly unstable (Bashirullah et al.,
1999
) (R. L. Cooperstock and H. D. Lipshitz, personal
communication). Another factor in favor of Hsp70Ab and Act5C
3'UTR is that the endogenous Hsp70Ab and Act5C genes
are indeed expressed in the wing imaginal disc cells; Hsp70Ab is
expressed in response to heat shock and Act5C is expressed
constitutively.
|
We analyzed the GFP expression from the two sets of reporter genes (UGA and UgGA, and UGH and UgGH) that are expected to express broadly, with an aim to compare effects of the insertion of a generic intron and the two different trailers. The GFP fluorescence was analyzed in third instar larval fillet preparations from which the autofluorescing gut and Malpighian tubules were removed. UGA and UgGA expressed GFP broadly in all larval tissues as expected from the Ubip63E promoter (Fig. 3A, data for UGA not shown). The expression from these transgenes was similar, showing that the generic intron is spliced efficiently in all tissues. However, in UGH and UgGH larvae, a weaker GFP signal was observed in most tissues, but a strong expression was seen in the brain and salivary glands, and a very strong signal was seen in male gonads (Fig. 3B, data for UGH not shown). Again, both UGH and UgGH expressed similarly. Although we analyzed all the four transgenes throughout these studies, we show results from only UgGA and UgGH, as UGA behaved similar to UgGA and UGH behaved similar to UgGH in all our experiments.
|
The differences between the expression of transgenes with the Hsp70Ab 3'UTR (UGH, UgGH) and the Act5C 3'UTR (UGA, UgGA) are noteworthy as the two sets of transgenes differ only in their 3'UTR. The non-uniform distribution of GFP signal in the UgGH larvae could be due to differentially higher levels of transcription, stabilization, degradation, and/or translatability of the mRNA selectively in certain tissues. Since UgGA does not show a similar differential expression pattern, the possibility of higher levels of transcription is unlikely. These observations are consistent with the relative higher stability of mRNAs with Act5C 3'UTR.
Next, we analyzed the expression from transgenes requiring neural-specific splicing for GFP synthesis. In UnGA larvae, the nervous system cells showed a bright GFP signal, although a weak but discernable signal was seen in male gonads (Fig. 3C). Additionally, a very low basal level of expression that is barely above background, cannot be ruled out in most other tissues (Fig. 3C). In UnGH larvae, a very bright signal was also seen in male gonads in addition to the nervous tissue, and again, a just above background signal cannot be ruled out for many other tissues (Fig. 3D). The neural expression of UnGA and UnGH is expected because the nASI splicing, which requires ELAV, should occur only in neurons; however, high levels of expression in UnGH male gonads is surprising as ELAV is not expressed in male gonads. We expect that the selective high signal in male gonads with UgGH and UnGH is due to the specific RNA processing molecules that probably exist within these cells. The very low level of general expression of UgGH and UnGH must be due to some baseline neural splicing occurring in all cells.
We also analyzed the expression of both sets of transgenes in the wing imaginal discs, as we planned to use that tissue for our ectopic expression studies. In the wing disc, uniform low level GFP expression was seen for UgGA and UgGH (Fig. 4I,L). GFP signal observed in the wing discs of UnGA or UnGH was extremely low, but discernable when compared with the control disc without transgenes (compare Fig. 4C,F with A).
|
In contrast to ELAV, ectopic HuD expression does not effect
neural-specific intron splicing in wing disc cells
To determine functional similarities and differences between ELAV and HuD,
we studied their effects on the GFP reporter transgenes described above. To
express ELAV and HuD in wing imaginal disc, we chose dpp-GAL4 driver,
as it expresses in wing discs in a stereotypic pattern
(Staehling-Hampton et al.,
1994). The expression pattern of dpp-GAL4 visualized
using UAS-GFP in wing disc cells shows a wide band of cells that runs
in the middle along the dorso-ventral axis
(Fig. 4B).
We examined wing discs ectopically expressing HuD and ELAV as described in
Materials and Methods. Previous studies have shown that ELAV is sufficient in
the wing disc cells for neural splicing of nrg, ewg and arm,
its three identified targets (Koushika et
al., 2000); however, whether ectopically-expressed HuD will also
influence neural splicing of the nASI was not known. As expected, the
control ELAV-expressing wing discs show GFP signal along the GAL4-expressing
band of cells from both UnGA and UnGH transgenes
(Fig. 4E,H). In contrast, in
HuD-expressing wing discs, the signal was uniformly low with either
UnGA or UnGH (Fig.
4D,G) and comparable with controls when there was no HuD ectopic
expression (Fig. 4C,F). Thus,
ectopic expression of HuD does not induce neural specific splicing.
Previously, using a different GAL4 driver we have demonstrated that
ectopically-expressed ELAV was sufficient to induce the neural protein isoform
of endogenously expressed genes nrg and ewg in wing disc
cells (Koushika et al., 2000).
In that system, ectopically-expressed HuD was also unable to generate neural
protein isoforms, Nrg180 or 116-kDa EWG (data not shown). Thus, HuD
fails to induce neural isoforms, in Drosophila wing disc cells, of
either the endogenous genes, nrg or ewg, or the GFP reporter
genes in which the splicing of nASI is essential for reporter
expression.
Both ELAV and HuD enhance GFP signal from transgenes with
Hsp70Ab 3'UTR
Next we asked whether the ectopic expression of HuD and/or ELAV influences
the level of GFP from the intron-less transgenes, or those with generic
intron. This could happen as a consequence of stabilization of the transcript
by HuD and/or ELAV. Both UgGA and UgGH cause relatively
uniform expression in the wing disc cells
(Fig. 4I,L). Wing discs
ectopically expressing HuD or ELAV were examined. In UgGA wing discs,
ectopic expression of HuD or ELAV still resulted in a uniform GFP expression
(Fig. 4J,K). In contrast, in
UgGH wing discs, ectopic expression of either HuD or ELAV resulted in
strong enhancement of the GFP signal along the dpp-GAL4 expression
band (Fig. 4M,N). The enhanced
signal was somewhat stronger with HuD than with ELAV. Since UgGA and
UgGH transgenes differ only in the 3' trailer, the enhancement
of GFP signal specifically seen in UgGH is likely to be due to its
Hsp70Ab trailer. The enhanced GFP signal could result from increased
stability or translatability of the transgene generated message. Given that
HuD has been shown to stabilize transcripts, we suggest that, in the wing
discs, both HuD and ELAV can stabilize mRNAs with the Hsp70Ab
trailer, perhaps through its ARE-like sequence.
In situ RNA analysis of transgenes UgGA and UgGH
with ectopically expressed ELAV and HuD
To determine if the enhanced GFP signal in ELAV- or HuD-expressing wing
discs is accompanied by increased transcript level, in situ hybridization was
performed as described in Materials and Methods. GFP transcript of
UgGH was compared with UgGA in wing imaginal discs that also
ectopically expressed ELAV or HuD. As shown in
Fig. 5, the expression of HuD
and ELAV resulted in a higher transcript signal in the band of GAL4-expressing
cells in UgGH wing discs (Fig.
5C,D), but not in UgGA wing discs
(Fig. 5A,B). This result is
consistent with the notion that ELAV and HuD stabilize mRNAs with
Hsp70Ab 3'UTR. However, selective increased transcription from
the UgGH transgene somehow facilitated by the Hsp70Ab
3'UTR only when ELAV or HuD are expressed remains a possibility.
|
Localization of ectopically expressed ELAV and HuD in wing disc
cells
We were interested to see whether ectopically-expressed ELAV in the wing
disc cells is localized mainly in nuclei, similar to endogenous or
overexpressed localization in neurons, or whether it showed perturbed
localization. To analyze the localization of ectopic ELAV and HuD, wing discs
expressing ELAV or HuD were immunostained using anti-ELAV or anti-HuD
monoclonal antibodies. The samples were analyzed by confocal microscopy after
propidium iodide staining to visualize the nuclei. ELAV expressed in the wing
disc cells was localized to both the cytoplasm and the nucleus, with a robust
signal in the cytoplasm (Fig.
6A-C). This is a clear departure from the mainly nuclear
localization of endogenous ELAV in neurons or even overexpressed ELAV in
neurons. HuD in wing disc cells was localized predominantly to the cytoplasm
(Fig. 6D-F) similar to what is
seen in the vertebrate cell cultures
(Wakamatsu and Weston, 1997)
and in Drosophila neurons
(Yannoni and White, 1999
). The
difference in localization of ectopically expressed ELAV in non-neural cells
could potentially explain its role in stabilizing the Hsp70Ab
3'UTR-containing RNAs. Conversely, the inability of HuD to splice the
nASI, could be largely due to the cytoplasmic localization of
HuD.
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Discussion |
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Hu proteins have been shown to bind ARE sequences through RRM domains
(Chung et al., 1996;
Levine et al., 1993
). The
structural similarities between the RRM domains of ELAV and HuD predict that
they ought to share RNA recognition specificity, as the residues that contact
RNA are conserved between the first two RRM domains with the exception of a
single conservative substitution (Wang and
Tanaka Hall, 2001
). Furthermore, the RRM3 domain of HuD, and that
of its closely related Drosophila homologue RBP9, can fully
functionally substitute the RRM3 domain of ELAV using viability rescue of a
null allele as a criterion (Lisbin et al.,
2000
). Thus, the RRM domains in the two proteins could be expected
to behave similarly. However, functional tests on chimeric proteins with RRM1
and RRM1RRM2 from RBP9 yielded rescue values of about 6% for RRM1
substitution, while no rescue was obtained with a RRM1RRM2 substitution,
suggesting that other amino acids in addition to RNA contact residues are
required for function (Lisbin et al.,
2000
), as these were precise RRM substitutions in the context of
ELAV protein.
Ectopically expressed ELAV and HuD enhanced GFP signal from UGH or UgGH transgenes, but not UGA or UgGA transgenes. The increase in GFP signal was accompanied by an enhancement of transcript level. A straightforward explanation is that Hsp70Ab 3'UTR is acting as a HuD-responsive element and that HuD is acting true to its documented function in the mammalian cells. An alternative possibility that there is selective increased transcription from the transgenes with Hsp70Ab 3'UTR when ELAV or HuD are expressed is unlikely but cannot be formally ruled out. Furthermore, the increase in transcript level suggests that increased translatability of the mRNA is not the likely cause of enhancement of GFP signal.
That ELAV mimics HuD in the wing disc cells raises an intriguing question. Does ELAV also stabilize certain transcripts in neural cells? Strong GFP expression in the central nervous system observed in UGH and UgGH larvae (Fig. 3B, data for UGH not shown) could be explained by endogenous ELAV which stabilizes the GFP transcript. We tried to address this issue by overexpressing ELAV in the nervous system and assessing the GFP signal from the UGH transgene with several GAL4 drivers (data not shown). No enhancement was observed. We believe that in Drosophila neurons UGH signal is not further enhanced by overexpression of ELAV, with a caveat that a slight enhancement would not have been necessarily discerned in this assay.
Several arguments led us to speculate that Hsp70Ab 3'UTR
behaves as an ARE in this assay. First, both HuD and ELAV have been shown to
interact with A/U-rich sequences (Chung et
al., 1996; Lisbin et al.,
2001
; Park et al.,
2000
), and Hu proteins have been shown to stabilize mRNAs
containing AREs (reviewed by Brennan and
Steitz, 2001
; Keene,
1999
). Second, Hsp70Ab 3'UTR has potential ARE-like
composition and it is unstable at non-heatshock temperatures. If
Hsp70Ab 3'UTR is indeed acting as an ARE-like element, it
suggests that Drosophila cells possess the molecular components
necessary for the HuD-mediated mechanism for ARE-related turnover and could be
used to study this process in vivo. This is consistent with the idea that the
mechanism of ARE-mediated mRNA turnover is conserved from yeast to humans
(Vasudevan and Peltz,
2001
).
Our studies have focussed on the role of ELAV in regulated neural-specific
splicing in Drosophila neurons. We first identified nrg as
an ELAV-regulated gene by demonstrating that ELAV is essential in neurons and
is sufficient in non-neural wing disc cells for the formation of a
neural-specific isoform of Nrg (Koushika
et al., 1996). The interpretation of this finding as ELAV being
required for neural-specific spliceform generation was subject to the
criticism that it could also be explained by selective stabilization of the
neural-specific transcript or increased stability of protein. Subsequent
studies on nrg splicing and on the splicing of ewg, a second
downstream target of ELAV, have reinforced the role of ELAV in splicing
regulation. These follow-up studies demonstrated: (1) a requirement of
ewg introns for ELAV-mediated regulation
(Koushika et al., 2000
); (2)
similar tissue-specificity for expression of reporter gene constructs that
report on ELAV-mediated splicing with several distinct 3'UTRs
(Lisbin et al., 2001
); and (3)
ELAV-binding sites within the nrg regulated intron, nASI
(Lisbin et al., 2001
). The
current study provides further convincing evidence that the generation of
neuron-specific isoforms is at the level of splicing. First, HuD expression
leads to the stabilization of UgGH transcripts, yet fails to induce
UnGH expression. Second, ELAV expression does not affect the level of
UgGA expression, but still induces UnGA expression in a
non-neural tissue. Together, these results show that the basal level
production of the neural-specific spliceform in non-neural wing disc cells is
not sufficient to induce expression through selective mRNA stabilization, and
that HuD appears to be lacking the ability to mediate neural splicing in these
cells. These findings underscore the functional divergence of these two
proteins in these cells.
In this study, stabilization function correlates with cytoplasmic
localization and splicing correlates with nuclear localization of ELAV and
HuD. Both ELAV and HuD appear to be comparably expressed in the cell, as
assessed by their effect on UGH and UgGH expression, and
both are expressed in the cytoplasm, but only ELAV shows significant
localization in the nucleus. Thus, the inability of HuD to mediate splicing
could be explained by its mainly non-nuclear localization, allowing the
possibility that if localized to the nucleus it could mediate splicing.
Consistent with this finding are our previous results with certain ELAV mutant
proteins that localize mainly to the cytoplasm in neurons. These proteins are
unable to provide ELAV's vital function, but their function is partially
restored when forced into the nucleus by tagging them with an exogenous
nuclear localization signal (Yannoni and
White, 1999). We envision that inherent differences between ELAV
and HuD result in differential interactions with factors that they encounter
in the wing disc cells. These different interactions contribute to the
differences in function and localization. For example, recently several
proteins that associate with HuR have been identified; at least two of these
have been implicated in nuclear export of HuR
(Brennan et al., 2000
;
Gallouzi et al., 2001
).
Regulation of alternative RNA splicing and mRNA stability are key processes that influence qualitative and quantitative aspects of proteins produced from a gene. Individual ELAV/Hu-family proteins have evolved to control these and other regulatory steps. This study signifies the importance of cellular context and the proteins that ELAV/Hu-family proteins associate with to the specificity of its function and suggests that evolution has tailored ELAV-family proteins along with the cellular context for specific post-transcriptional processes.
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Acknowledgments |
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References |
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