RESEARCH PAPER
RESEARCH PAPER
RNA Biology 10:8, 1396–1406; August 2013; © 2013 Landes Bioscience
Half Pint/Puf68 is required for negative regulation
of splicing by the SR factor Transformer2
Shanzhi Wang,1,2,3 Eric J Wagner2,4, and William Mattox1,2,3,*
1
Program in Genes and Development; The University of Texas Graduate School of Biomedical Sciences at Houston; Houston, TX USA; 2The University of Texas Graduate
School of Biomedical Sciences at Houston; Houston, TX USA; 3Department of Genetics; University of Texas M.D. Anderson Cancer Center; Houston, TX USA; 4Department of
Biochemistry and Molecular Biology; University of Texas-Houston Medical School; Houston, TX USA
Keywords: RNA splicing, Transformer2, Half pint, splicing repression, Drosophila
The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of
organisms. These factors affect splicing through both positive and negative controls of splice site recognition by prespliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses
splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator
region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferasebased splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This
approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of
the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and
that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient
to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68,
and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of
the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while
Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and
suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these
studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2dependent intron inclusion.
Introduction
Alternative splicing of pre-mRNA plays a vital role in producing
a flexible and complex diversity of proteins from a more fixed
genome. Transcriptome analyses indicate that a majority of genes
in metazoans produce alternative transcripts.1,2 Although a large
number of factors affecting alternative splicing have been found,
the mechanisms by which these factors interact with pre-mRNA
to alter splicing in developmentally specific patterns are still
under investigation. Moreover, the mechanisms by which these
factors control splice site selection are not completely understood.
The serine/arginine (SR)-rich proteins and SR-related factors
play important roles in the regulation of alternative pre-mRNA
splicing. Numerous studies have now documented the ability of these RNA binding proteins to activate alternative splice
sites through interactions with nearby exonic splicing enhancers (ESEs) (for review, see ref. 3). SR-ESE complexes activate
splicing through the recruitment and stabilization of pre-spliceosomal complexes at the alternative splice site, committing the
pre-mRNA to selection of the activated site.4 SR proteins bind
to ESEs through their RNA recognition motifs (RRMs) and
facilitate pre-spliceosome formation through interactions of their
Arg-Ser-rich effector domains.5-11
While activation of splicing through binding to ESEs has
been widely observed, it is not the only function of SR proteins
in the regulation of splicing. In some transcripts, SR factors have
been found to negatively regulate splice site recognition.12-15 The
Transformer 2 (Tra2) protein, a component of the Drosophila sex
determination pathway and one of the founding members of the
SR protein family, provides a case in point. When bound to ESE
elements within an exon of the doublesex (dsx) pre-mRNA, Tra2
robustly activates a nearby sex-specific splice site by forming an
enhancer complex with Transformer (Tra) and SR factors.16-18
This complex recruits U2AF to the splice site and facilitates prespliceosome formation. In contrast, Tra2 also binds directly to
regulatory elements within the M1 intron of its own pre-mRNA
where it inhibits pre-spliceosome assembly and causes repression of splicing through intron retention.13,14 Similar inhibitory
functions of SR factors have also been observed in mammalian
cells.12,19,20
Recent findings indicate that the effect of splicing factors on
splice site recognition is highly dependent on the position where
*Correspondence to: William Mattox; Email: [email protected]
Submitted: 04/15/2013; Revised: 06/21/2013; Accepted: 07/05/2013
http://dx.doi.org/10.4161/rna.25645
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RESEARCH PAPER
machinery.24 However, the nature of the latter interactions and
the specific cofactors involved have not yet been determined.
To better understand how Tra2 represses splicing of its M1
intron, we performed an RNAi-based screen in search of corepressors. Among the factors identified using this approach was
Half pint (Hfp), a conserved protein that has previously been
shown to play roles in the regulation of alternative splicing as
well as in the direct transcriptional regulation of the myc oncogene in both flies and mammals.28-30 We find that Hfp is required
for repression of M1 splicing by Tra2 both in cultured cells and
within the male germline in vivo, but is not required for Tra2dependent activation of dsx splicing. Moreover, we report the
expression of alternative forms of the Hfp protein that differ in
their ability to repress M1 splicing uncovering an additional layer
of regulation of Tra2-dependent splicing repression. Collectively,
these results illustrate that differing Tra2 activities are mediated
through unique interactions with distinct cofactors.
Results
Figure 1. A luciferase reporter detects specific repression of M1 splicing. (A) The organization of reporter plasmids used to screen for Tra2
cofactors in Drosophila S2 cells is shown. Splicing of the M1 intron from
pM-Luc transcripts leads to expression of firefly luciferase (Luc). The
light gray exons and M1 intron derive from the endogenous Drosophila
Tra2 gene. The initiation codon shown is naturally split by the intron
and is in frame with luciferase coding sequences. The ISS element is
indicated within M1 intron. Transcript from pftz-Luc contains exons
and a constitutive intron from the Drosophila ftz gene. The same
promoter and polyA signals are used in both constructs. (B) Luciferase
activity from pM-Luc but not pftz-Luc is repressed in the presence of
pT2 a plasmid expressing the Flag-tagged Tra2-PC isoform of Tra2 in
cotransfection experiments. Results from luciferase assays (graph) and
immunoblots probed with anti-Flag antibodies are shown. The position
of Flag-Tra2 and two non-specific bands (nc) typically observed in such
assays as well as molecular weight markers are indicated. (C) The effects
of Tra2 on splicing of transcripts from both reporters, as detected by
RT-PCR, is also shown. As expected, the ratio of amplification products
from unspliced (U) to those of spliced (S) transcripts deriving from pMLuc increases with higher levels of Tra2.
the protein binds relative to the affected splice site.21-24 In the
studies of Tra2, it was observed that Tra2 bound at intronic positions elicits splicing repression, but binding at exonic positions
causes splicing activation.25,26 Moreover, tethering experiments
show that while activation of splicing is mediated by effector
functions of the Arg-Ser-rich region of the protein, repression
depends on an effector function intrinsic to the RRM-linker
region.26,27 This suggests that these opposing functions are performed through distinct molecular interactions with the splicing
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Half-pint is required for Tra2-dependent repression of M1 splicing. To identify factors that contribute to Tra2-dependent splicing repression, we designed a reporter plasmid (pM-Luc) in which
the expression of firefly luciferase depends on splicing of the M1
intron (Fig. 1A). This reporter plasmid contains the intact M1
intron as well as exons 3 and 4 positioned upstream of a firefly
luciferase open reading frame. The intron includes the intronic
splicing silencer (ISS) sequences previously shown to be sufficient
for both binding of Tra2 and repression of splicing in nuclear
extracts.14 A naturally occurring translation initiation codon is
split by the M1 intron and is positioned as the only initiator that
is in-frame with luciferase coding sequences. As a control, we created an analogous reporter using the ftz exons 1 and 2, which
has been shown previously to be recalcitrant to Tra2 regulation.13
Transfection of either reporter into S2 cells resulted in efficient
and detectable luciferase expression (Fig. 1B). However, cotransfection of these reporters into Drosophila S2 cells along with a
plasmid (pT2) that express Tra2 resulted in a specific reduction
in luciferase (Fig. 1B) from the M1 intron containing reporter
in a dose-dependent fashion. No repression was observed for the
control pftz-luc reporter (Fig. 1C). Semi-quantitative RT-PCR
analysis of RNA isolated from transfected cells revealed a correlative increase in the levels of unspliced pM-Luc-derived mRNA,
while no change in splicing was observed for the control pftz-luc
mRNA. These results show that the pM-Luc reporter is a specific
indicator of Tra2-dependent splicing repression.
Given these proof-of-principle results, we next tested the effect
that individual dsRNA knockdowns of 247 factors with known
or potential roles in RNA metabolism31 had on pM-Luc splicing
in the presence of Tra2. Luciferase assays on these knockdowns
identified a group of 10 candidate factors that exhibited larger
reductions in pM-Luc activity than did parallel positive controls
in which Tra2 protein levels were reduced with dsRNA (Fig. S1).
Among these candidates, the Half-pint (Hfp) protein, also
known as Puf68, was selected for further analysis as it has previously been implicated in the regulation of alternative splicing.30,32
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To address any potential off-target effects of the RNAi, we first
tested whether a second dsRNA targeted at Hfp also produced
significant effects on pM-Luc splicing in the presence of Tra2.
Western blot analysis confirmed that, like the original dsRNA
tested (Fig. 2C), this dsRNA was effective in depleting Hfp protein levels and derepressed luciferase activity from the reporter
(Fig. 2A). This demonstrates that Tra2-mediated repression
indeed requires Hfp expression. As expected, RT-PCR experiments showed that the higher luciferase activity was indicative
of an increase in M1 splicing from the reporter (Fig. 2B) confirming that knockdown of Hfp impairs M1 repression. We also
examined whether the effect on M1 splicing might result from
an indirect effect on the amount of Tra2; however, we found
that the level of plasmid-derived protein was unchanged by Hfp
knockdown (Fig. 2C). Taken together, the above results indicate that Hfp participates in the repression of M1 splicing in
this system.
Half-pint associates with Tra2 transcripts. To determine
if Hfp physically associates with Tra2 transcripts, we next performed RNA immunoprecipation experiments with anti-Hfp
antibody from S2 cell lysates cotransfected with three separate
reporters, pM-Luc, pftz-Luc, and pAct-GFP (Fig. 3A). The
precipitated material was then subjected to RT-PCR with primers spanning the intron and flanking exons to detect associated
RNA sequences. As shown in Figure 3A and Figure S2, specific association of Hfp was observed with both the spliced and
unspliced transcripts of pM-Luc, but was not found to associate with pftz-Luc or pAct-GFP transcripts. Furthermore, Hfp
was found to associate with endogenous Tra2 in S2 cells. Again,
association was observed in transcripts both with and without
the M1 intron (Fig. S3). Notably in both pM-Luc and endogenous transcripts containing M1 were preferentially precipitated
over those lacking the intron. Taken together, these results indicate that the association of Hfp depends on sequences located
both within and outside of the M1 intron with the stronger associations specified by intron sequences.
As Tra2 is known to repress splicing by binding to the ISS
sequences within the M1 intron, we also tested whether the
interaction of Hfp with pM-Luc transcript was affected when
levels of Tra2 were increased (see +pT2 in Fig. 3B). These
experiments revealed a small but consistent increase in Hfp
binding in lysates from pT2-transfected cells for all three amplicons spanning unique regions with the pM-Luc reporter. The
small increase in binding signals observed could be due to the
increased M1 retention that increases the amount of the proteins
preferred target.
Previous crosslinking studies on Tra2 indicated that it binds
ISS sequences with moderate affinity and high specificity in the
presence of Drosophila nuclear extracts, but fails to do so in the
absence of complementing extracts.13,14 We therefore next examined whether Hfp alone can bind directly to an RNA containing
the M1 intron and it can facilitate Tra2 binding in the absence of
extract. Gel shift assays (Fig. S4 lane 3 and Fig. S5) performed
with low micromolar concentrations of recombinant Hfp protein
produced an shift in the mobility of RNA containing the entire
M1 intron suggesting that the protein can bind RNA directly
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Figure 2. Half pint is required for repression of M1 splicing in pM-Luc
transcripts. (A) The effect of 2 μg and 6 μg of Hfp dsRNA on luciferase
activity from Drosophila S2 cells cotransfected with pT2 and pM-Luc is
shown. The graph shows percent luciferase activity in relation to cells
transfected with an empty expression vector. Hfp68 and tubulin protein
levels from the same cells are analyzed in the western blot below the
graph. All samples are transfected with the same amount of pM-Luc.
Error bars represent SD values (n = 3). (B) The ratio of unspliced to spliced
pM-Luc transcripts as determined by qRT-PCR of cells treated with Hfp
dsRNA is shown in the graph. Error bars represent SE values (n = 2).
Primers positions are indicated by arrows in the diagram. A gel showing
unspliced (U) and spliced products (S) from a parallel semi-quantitative
PCR analysis with different primers is shown below the graph. (C) western blots detecting expression of Flag Tra2 from cells transfected with
pT2 and endogenous Hfp after treatment with Hfp dsRNA and control
dsRNAs are shown. In all experiments, a monoclonal anti-Hfp antibody
was used to detect level of Hfp. The M2 monoclonal anti-Flag antibody
was used to detect transfected Flag-Tra2.
with at least low affinity. Addition of Tra2 and Hfp proteins
together (Fig. S4, lane 5) led to the appearance of a new complex with lower mobility than obtained with either protein alone.
Together, these results suggest that Hfp and Tra2 are capable of
co-occupying the M1 intron and that associations observed in
vivo are likely to be further facilitated by additional unknown
factors.
The effect of Hfp on splicing depends on the M1 ISS and
a suboptimal 3' splice site. We next tested whether sequences
previously found to support binding and repression by Tra2 are
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Figure 3. Half pint is associated with Tra2 pre-mRNA. (A) RNA-protein
complexes from S2 cells cotransfected with equimolar amounts of pMLuc and two control reporter constructs were immunopreciptiated with
anti-Hfp antibodies and analyzed by RT-PCR. Primer positions for each
reporter are diagrammed to the left and products are shown to the right.
Lanes are loaded with products amplified from 20% of input lysate (In),
eluate from beads only (BO) and eluate from beads coated with anti-Hfp
antibody (BA). The three reporter constructs contain the same promoter
and polyA signal sequences. (B) Similar RNA immunoprecipitation assays
were performed with anti-Hfp antibody on various regions near or within
the M1 intron of pM-Luc after cotransfection with and without pT2. The
numbered primers correspond to the individual immunoprecipitations
as indicated. Products from amplification of 50% input (In), eluate from
beads only (BO) and eluate from beads with antibodies (BA) are shown.
also sufficient for the observed effects of Hfp.14,33 As illustrated
in Figure 4A, the native M1 intron contains two conserved
sequences known to be important in M1 splicing. Based on
assays in S2 nuclear extracts, the 80 nt ISS alone was found to
be sufficient to confer Tra2-dependent repression of splicing on
an intron that is otherwise recalcitrant to regulation by Tra2.14,26
Furthermore, experiments performed in transgenic flies determined that the intron’s weak 3' splice site context is also required
for repression by Tra2.33 Notably, a weak 3' splice site from the
myosin heavy chain gene (mhc) is sufficient to replace the native
weak M1 sequences, suggesting this requirement is general and
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not specific to the native Tra2 3' sequences. The strong 3' splice
site from the ftz gene was also found not compatible with splicing repression in vivo.33
To determine if the ISS and a weak 3' splice site are sufficient
for repression by Hfp in a manner similar to Tra2, a series of
splicing reporters based on pftz-Luc was constructed in which
expression depends on splicing of the ftz intron (Fig. 4A).
Each of these splicing reporters was then cotransfected with
increasing amounts of Tra2 and relative luciferase activity
was then measured. We observed that when a combination of
the ISS and the mhc weak 3' splice site (ftz-ISS-mhc-Luc) are
substituted into this intron they are sufficient to allow dosedependent repression of reporter activity by Tra2 (Fig. 4B). In
contrast, constructs missing either of these individual elements
failed to undergo significant repression in response to elevated
Tra2. These results confirm that the ISS and weak 3' splice site
are both required for Tra2-dependent splicing repression in S2
cell transfections as inferred from other systems.
To determine if the pftz-ISS-Mhc luciferase reporter requires
Hfp expression for Tra2-mediated splicing repression, we
repeated the transfections under conditions that deplete Hfp.
Notably, the knockdown of Hfp led to a significant reversal
of the Tra2-induced repression when the ftz intron contained
both the ISS and the weak Mhc 3' splice site (Fig. 4C). These
results are consistent with the data shown in Figure 2A and also
strengthen the conclusion that Hfp is a cofactor for Tra2 as a
heterologous reporter sensitive to Tra2 also requires Hfp expression for full splicing repression. Moreover, knockdown of Hfp
also led to a similar reversal of both pM-Luc and ftz-ISS-MhcLuc (Fig. 4D) when tested in the presence of only endogenous
Tra2. Together, these results indicate that Hfp affects splicing
through interactions with the ISS in cooperation with Tra2.
Hfp is required in vivo for repression of M1 splicing but
not for activation of an alternative splice site in dsx premRNA. In Drosophila development, repression of M1 splicing
is most prominently observed in the male germline where about
50% of Tra2 mRNA retains the M1 intron.34 To determine if
Hfp plays a significant role in splicing repression in vivo we
dissected testes from wild-type and hfp mutant adults. Because
strong loss of function results in lethality at earlier stages, this
analysis was performed with hypomorphic hfp alleles that allow
adult survival.30 As shown in Figure 5A, endogenous M1 splicing efficiency was found to be significantly increased when
Hfp function was reduced. For comparison, a parallel RT-PCR
assay with RNA from tra2 mutant testes is shown in which M1
splicing is also increased. These results demonstrate that Hfp is
required for repression of M1 splicing in the male germline.
We next tested whether the female-specific splicing of the dsx
pre-mRNA, which is activated by Tra2, also depends on Hfp
function. Splicing was analyzed in wild-type, tra2, and hfp
mutant adult flies by RT-PCR performed on total RNA with
primers that detect the male and female-specific dsx transcripts
as distinct products diagrammed in Figure 5B. The results of
this experiment, shown in the lower part of Figure 5B, indicate
that Hfp loss of function did not reduce the activation of the
Tra2-dependent female-specific dsx 3' splice site. Thus, Hfp is
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Figure 4. Repression of splicing by Hfp and Tra2 depend on both the
ISS and a suboptimal 3' splice site. (A) The diagram shows various
constructs used to test the specificity of Hfp for the ISS and a weak 3'
splice site. The organization of the Tra2 M1 intron (top) is shown only
for reference. All experiments were performed in constructs based on
the ftz intron (thin line) and its flanking exons (gray boxes). Thickened
lines indicate the positions where the ISS and the polypyrimidine
tract/3' splice site. The ftz intron naturally has a strong 3' splice site and
no ISS. In ftz-mhc, a weak 3' splice site of mhc intron 5 is substituted
into this intron, in ftz ISS the Tra2 ISS is substituted, and in ftz-ISS-mhc
both are present in combination. (B) Luciferase assays performed after
cotransfection with different amounts of pT2. Significant repression is
only observed with the native M1 intron (n = 7) and in ftz-ISS-mhc (n =
9), but not in ftz (n = 7), ftz-ISS (n = 6), and ftz-mhc (n = 10), compared
with reporter only in each group. Error bars represent SD values. (C)
Knockdown of Hfp in cells transfected with pT2 derepresses luciferase
expression from ftz-ISS-mhc (n = 3). The graph shows relative luciferase
levels from treatment of cells various dsRNAs. Significant derepression
(*) was observed only with dsRNA directed at Tra2 and Hfp, compared
with pT2 transfected only. Error bars represent SD values. (D) RNA
interference with dsRNA directed at endogenous Hfp and Tra2 in S2
cells. Knockdown of Hfp significantly increases luciferase from the M1
and ftz-ISS-mhc reporter but not from other reporters, compared with
reporter only group (n = 7–10). Error bars represent SD values.
not generally required for all Tra2-dependent alternative splicing
events but rather is a specific cofactor required in the regulation
of M1 splicing.
Alternative forms of Half pint differ in their effects on M1
splicing. Analysis of the Drosophila transcriptome indicate that
it contains at least nine alternatively spliced mRNAs from the
hfp gene.35 These mRNAs are predicted to encode either of two
protein forms that differ by the presence of a 92 amino acid
N-terminal sequence, which contains four arginine-serine dipeptides (Fig. 6A; Fig. S6). Type A transcripts encode a 68 kDa
protein (Puf68) predicted to initiate translation in the first exon
and consistent with the major Hfp product previously reported.30
All other Hfp transcripts contain in-frame stop codons a short
distance downstream of this initiation site. However, initiation at
a downstream start codon found in these transcripts could potentially produce a 58 kDa protein (Fig. 6A; Fig. S6). Consistent
with this prediction, immunoblots performed on Drosophila
lysates with Hfp monoclonal antibody directed to the protein’s C
terminus detect both 68 and 58 kDa proteins (Fig. 6B and C; Fig.
S7). Importantly, both bands were reduced in level in flies with a
hemizygous hfp mutant genotype (Fig. 6B). In S2 cells, Hfp68 is
the naturally expressed form with much higher level than Hfp58
(Fig. 7A). The longer 5'UTR of Hfp58 transcripts, compared
with that of Hfp68, might be responsible for its low expression
level in cells. However, expression of either Hfp58 (Fig. 6C) or
both isoforms (Fig. 7A) was found to be increased when the corresponding hfp cDNAs were transfected into Drosophila S2 cells,
as expected if they are products of hfp mRNA. Notably, these
products also showed sex and tissue specificity with the 68 kDa
product being preferentially expressed in males and 58 kDa in
females (Fig. 6C). The gender bias of expression also suggests
that protein degradation is not the source of the lower product.
To test if these isoforms differ in their ability to support repression of M1 splicing, we knocked down endogenous Hfp protein
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using a dsRNA targeted to its 3'UTR (hfp-3'UTR dsRNA).
These cells were cotransfected with plasmids expressing either of
the Hfp isoforms from mRNA with a distinct 3'UTR sequence
as well as the pT2 and pM1-Luc plasmids. As shown in the
immunoblots in Figure 7A, endogenous Hfp was effectively
reduced by treatment with a dsRNA targeting the Hfp mRNA
3' UTR (Fig. 7A, lane 1 and 2) and the levels of luciferase activity increased due to reduced M1 intron retention. As expected,
expression of Hfp68 restored luciferase activity to nearly its original levels (Fig. 7A, lanes 5 and 6), but unexpectedly, expression
of Hfp58 did not reverse this effect (Fig. 7A, lane 3, 4). These
results suggest that Hfp68, but not Hfp58, is capable of mediating repression of M1 splicing. This conclusion is supported by
quantitative RT-PCR analysis of mRNA isolated from S2 cells
transfected analogously (Fig. 7B). The spliced/unspliced ratio
of pM-Luc transcripts was found to significantly increase upon
Hfp knockdown and to be restored after expression of Hfp68
but not Hfp58.
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by Tra2. This finding was initially based on an RNAi
screen performed in Drosophila cultured cells with a splicing reporter construct and was extended to living flies by
examining how loss of function mutations in Hfp affects
repression of M1 splicing in a developmental context where
it normally occurs. We observed that Hfp is required for
detection of intron retaining transcripts in mRNA derived
from male testes where Tra2 repression normally blocks
splicing of M1 in about half of Tra2 mRNA molecules
in response to a negative feedback mechanism.34 Splicing
repression in this tissue is thus dependent on both Tra2 and
Hfp.
Previous studies show that the dependence of M1 repression on the Tra2 protein in the male germline is complete.
In its absence, the intron is spliced from all detectable
germline mRNA.36 Thus, the endogenous Hfp present in
the male germline is unable to cause M1 repression in the
absence of Tra2. Whether Tra2 can cause repression independently of Hfp is presently unclear because only partial
loss-of-function mutations in Hfp are available and they
display low residual M1 repression.
The results presented here depend on the use of S2 cells
to study an alternative splicing event that normally occurs
primarily in the male germline. Although it is possible that
germline-specific splicing factors are needed in vivo for M1
Figure 5. Half pint loss of function affects M1 splicing but not dsx splicing
splicing repression, extensive in vitro analysis of splicing
in vivo. (A) RT-PCR analysis of M1 splicing in adult testis mRNA from various
has demonstrated that S2 nuclear extracts are competent
genotypes is shown. Primer locations are indicated by arrow the diagram. In
to support sequence-specific repression of M1 splicing by
w1118 flies that are wild type for both tra2 and hfp, the M1 intron is retained in a
majority of transcripts. Loss of function for either tra2 or hfp result in reduced
Tra2.13,14,26 Consistent with this, previous studies on transM1 retention. tra21 and tra2b are point mutations that do not alter the total
genic fly strains clearly showed that Tra2 has the potential
amount of Tra2 mRNA. Df(hfp) corresponds to Df(3L)AR148 is a large deletion
to repress M1 splicing in somatic cells in vivo when it is
that spans the entire hfp gene. (B) Similar RT-PCR analysis was performed on
expressed at high levels comparable to those normally
dsx RNA from whole adult males (M) and females (F) of various genotypes.
found in the germline.25 This observation is important as
Three primers, as indicated by arrows in the diagram, were used together to
amplify both male and female-specific splicing products of dsx. As expected
Tra2 transcripts are generated from a distinct promoter in
mature male mRNA was detected in RNA from a loss-of-function tra2 mutant
the male germline that is significantly more active than the
genotype, but was not found in females from either of two strong loss-ofsomatic promoter. The appearance of M1 retaining RNA
function hfp mutant genotypes tested.
through negative feedback on splicing in the male germline
is most likely to arise because Tra2 is transcribed at its highHfp68 and Hfp58 differ in their nuclear/cytoplasmic local- est levels there. Thus, the factors responsible for tissue specificity
ization. The Hfp58 isoform lacks sequences at the N terminus of this splice choice are thought to act at the level of transcription.
of Hfp68 that are of unknown function. Immunofluorescence Using the promoter from the Drosophila actin5C gene here, we
staining of endogenous Hfp in S2 cells (which primarily express have provided high levels of Tra2 in S2 cells sufficient to cause
Hfp68) indicated that most Hfp protein in these cells is localized significant levels of M1 repression.
to the nucleus (Fig. 8A, Endo-Hfp). To determine if Hfp58 and
It is worth noting here however that using a splicing reporter
Hfp68 are likely to differ in this regard, we used an anti-Flag anti- in S2 cells we also observed basal M1 retention that occurred
body to similarly stain S2 cells transfected with either Flag-Hfp58 without overexpression of Tra2. Notably, this basal repression did
or Flag-Hfp68 (Fig. 8A). This revealed that while Flag-Hfp68 not respond dramatically to knockdown of endogenous Tra2 prois predominantly nuclear (Fig. 8B), Flag-Hfp58 is distributed tein but was nonetheless sensitive to knockdown of endogenous
almost equally between the nucleus and cytoplasm (Fig. 8B). Hfp and its response to Hfp required the presence of both the
These results suggest that the reduced ability of Hfp58 to repress M1 ISS and a weak 3' splice site. These observations suggests that
M1 splicing is in part due to reduced localization to the nucleus.
while Hfp is required for repression of M1 induced by high levels
of Tra2, it also has the ability to elicit a low level of sequence-speDiscussion
cific repression of splicing that is independent of Tra2 in S2 cells.
Although Hfp plays a required role in Tra2-dependent splicThe results reported here indicate the Hfp/Puf68 protein of ing repression, it does not appear to be needed for the wellDrosophila is required for the negative regulation of splicing characterized function of Tra2 in activation of splicing through
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exonic splicing enhancers as illustrated by the
absence of any visible effect of Hfp mutations
on female sexual differentiation or on the sexspecific splicing of dsx mRNA. The failure to
affect dsx splicing is unlikely to be the result of
tissue-limited expression of Hfp, as this protein
is known to be widely expressed and functional
within a variety of somatic tissues such as the
imaginal discs in which regulation of dsx splicing normally occurs.28,37 Therefore, it is likely to
be available in such tissues but simply does not
participate in dsx splicing. Thus, Hfp behaves
as a specific co-repressor of Tra2 but lacks a discernible co-activator function.
Hfp has previously been reported to play a
role in the regulation of alternative splicing in
the Drosophila female germline, but the results
presented here are the first suggesting that Hfp
can contribute specifically to repression of splice
site recognition. Indeed, Hfp was previously
observed to be required to promote inclusion of
an alternative exon in transcripts from the ovarian tumor gene expressed in the adult female
germline.30 Moreover, studies on PUF60/FIR,
the human ortholog of Hfp, showed that it
binds to and activates 3' splice sites that have
suboptimal polypyrimidine tracts.38 PUF60
function in this regard is intertwined with that
of U2AF65, which generally promotes 3' splice
site recognition.39 In vitro studies show that
PUF60 can synergize with U2AF65 to promote
splicing and in some situations is able to functionally substitutes for this factor.38 Thus, both
Hfp and its human ortholog, PUF60, have previously been regarded as factors that promote
Figure 6. Hfp has two isoforms expressed in vivo. (A) The exon-intron patterns giving rise
rather than repress splice site recognition. Our
to various Hfp (Puf68) encoding mRNAs. The diagram was created based on the informafinding that Hfp performs a negative function
tion in Gbrowse. Boxes correspond to exons, lines to introns. Gray shaded regions are
and collaborates with Tra2 thus broadens the
predicted to be noncoding, orange box is predicted protein coding. Note that only the RA
range of effects on regulated splicing that this
transcript has the potential to encode the Hfp68 isoform, all other transcripts are predicted
factor has been associated with.
to encode Hfp58. The initiation codons and stop condons are indicated. (B) A comparison
of Hfp68 expression in lysates from a mixture of Drosophila male and female adult flies with
A connection between Hfp and Tra2 was
various genotypes is shown. Both forms are observed in w1118 adults (carrying two wild type
also found in studies on ATR dependent alternaalleles of hfp), but are both reduced in hfp13/Df(hfp). Both hfp13 and hfp9 are partial loss-oftive splicing of Taf1 transcripts in Drosophila S2
function mutations. (C) Expression of Hfp68 and Hfp58 in w1118 male (M) and female (F) adult
cells.32,40,41 These two factors were both among
flies is shown as detected by immunoblotting. For comparison, lysates from S2 cells (−) and
a small group of RNA binding proteins shown
S2 cells transfected with Flag-Hfp58 (F-Hfp58) were loaded on the same gel.
to be required to regulate splicing of mRNA
encoding the Taf1–3 and Taf1–4 isoforms from
the Drosophila Taf1 gene. The similar dependence of Taf1 on protein. The first evidence for this came from the observation
these two factors suggests that perhaps regulation occurs through that certain mutations in Tra2 disrupt repression but have little
a similar mechanism. However, it is not yet known whether Tra2 if any effect on splicing activation.42 More recent experiments
and Hfp alter Taf1 pre-mRNA splicing primarily through posi- in which various Tra2 protein sequences were tethered to target
tive or negative effects on the alternative splice sites.
RNAs through fusion to the MS2 coat protein and its cognate
The identification of a specific protein co-repressor for Tra2 binding sites showed that activation depends on effector funcis of particular significance in light of recent studies indicat- tions of the protein’s Arg-Ser rich regions, while repression is
ing that the known activation and repression functions of Tra2 mediated through effector functions within the RRM-linker
are encoded by distinct and separable effector regions of the region.26,43 This separation of functions supports the idea that
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Volume 10 Issue 8
non-sex-specific SR proteins and the femalespecific Transformer (Tra) protein which both a
required and limiting factor in vivo. Tra2, Tra,
and these SR factors are believed to activate splicing through the combined action of their ArgSer-rich domains, which directly facilitate the
assembly of pre-spliceosomal complexes at the
dsx 3' splice site. The results we have reported
here indicate that Hfp has properties expected
of a co-repressor that could instead take part in
functions specific to repressive effector domain
of Tra2. Consistent with this idea, the effects
of Hfp on splicing were found to depend on
the same intronic sequence elements in the M1
intron that mediate RNA binding and repression
of splicing by Tra2. This suggests a close collaboration between these factors and the possibility
that Hfp is recruited to the transcript upon Tra2
binding. Our immunoprecipitation data also
indicates that Hfp affects M1 splicing through
either a direct or indirect physical interaction
with the target transcript in the presence of Tra2.
Aside from Tra2 and Hfp the only other cofactor
known to affect M1 splicing is Rbp1, a non-sexspecific SR protein that is also a component of
the dsx enhancer complex.14,46,47 The dual role of
Rbp1 is not well understood, but curiously it does
not cooperate with Tra2 in binding to the ISS.14
In summary, our results suggest that Hfp directly
facilitates splicing repression by Tra2 in a manner
unique among known Tra2 cofactors.
A key question that remains is mechanistically
how Tra2, in cooperation with Hfp, and perhaps
other factors inhibit early steps in splicing. Insight
to this question comes from the recent observation that the direction of Tra2-dependent regulation of splicing depends on whether this protein
and its associated factors are bound at an intronic
or exonic position.26,43 When Tra2 is tethered to
Figure 7. The alternative N-terminal region of Half pint is required for repression of M1
RNA at different intronic positions, or is bound to
intron splicing. (A) Transfection experiments performed with pM-Luc splicing reporter
native ISS elements placed at different locations in
and constructs expressing Flag-Hfp58 or Flag-Hfp68. Partial loss of endogenous Hfp
the intron, the protein acts as a splicing repressor.
using a dsRNA targeted at the 3' UTR (hfp 3'UTR dsRNA) derepresses Luc expression. The
But when the ISS or tethering sites are placed at
ability of each Hfp isoform to restore repression was tested by transfection of the cells
with expression constructs containing 3' UTR sequence from SV40 that are not affected
exonic positions then Tra2 promotes activation of
by the dsRNA. Levels of Hfp58 and Hfp68 as detected by immunoblotting with Hfp is
splicing in the same system. Repression of splice
shown below the chart. The 92 kD band shown is a non-specific cross-reacting protein
sites by intronically positioned Tra2 and activation
from the same gel that is used to represent loading. (B) Quantitative RT-PCR analysis of
of splice sites by exonically positioned Tra2 both
total cellular RNA from cells treated as in (A) (n = 3). The ratio of spliced/unspliced pMresult in the same outcome—the exonic identity of
Luc transcripts was determined using the same primers as were used in Figure 2B. For
all panels, results that affected significantly from controls treated with hfp-3' UTR dsRNA
the bound sequences. In the case of the M1 intron
only are indicated (*). Error bars represent SE values.
repression of its splice sites by Tra2 and Hfp blocks
pre-spliceosome assembly26 and results in retention
distinct cofactors are likely to be involved in activation and of the intron within a larger exon. As the intron’s 3' splice site is
repression.
a weak match to the Drosophila consensus and the recognition of
Known Tra2 co-activators include a number of proteins weak 3' splice sites is a characteristic feature of Hfp’s mammalian
that bind cooperatively with Tra2 to form the dsx splicing ortholog, one possibility is that Hfp assists Tra2 in identifying
enhancer complex.18,44,45 Identified among these are several such sites for repressive interactions.
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RNA Biology
1403
In addition to the above mechanistic insights, we also report
here for the first time the detection of two distinct forms of the Hfp
protein that differ by the presence of an alternative N-terminal
region. These two forms differ functionally in both their nuclear/
cytoplasmic distribution and their ability to affect splicing with
the larger Hfp68 being most localized to the nucleus and displaying the stronger effects. These isoforms appear to arise through
alternative splicing of Hfp pre-mRNA itself. Thus, regulation of
splicing may occur at multiple levels to affect expression of targets of Hfp and Tra2. Notably, we have observed that the intracellular distribution of Hfp differs between various cell types,
and that males and females differ in their relative accumulation
of these two isoforms. The existence of multiple protein forms
is particularly interesting in light of the observation that, aside
from its role in splicing, Hfp has also been reported to play a
conserved and direct role in the transcriptional regulation of the
c-myc oncogene.28,29
Materials and Methods
Plasmids and primers. The plasmid pM-Luc was constructed
based on pBluescript SK+ vector (pSK+) by inserting amplified fragment containing the actin 5C promoter, intact Tra2
sequences from M1 intron and its two flanking exons, and firefly
luciferase protein coding sequences. An SV40 polyadenylation
signal derived from pFastback-Flag-Tra2 was inserted downstream of the luciferase termination codon. Primers are listed in
the Table S1. The plasmid pT2, is designed to express Tra2-PC,
an isoform that was genetically defined as sufficient for both
repression and activation of splicing.36 It was generated by amplifying sequences from pFastBac 3XFlag-Tra2-PC and then inserting these into pSK+ contaning Actin5C promoter and SV40 poly
A signal (pSK-AS).
pFastBac 3XFlag-Tra2-PC was made by inserting a synthetic
DNA fragment (Integrated DNA Technologies) encoding a
3XFlag tag at the N terminus of Tra2 PC coding sequence in
pFastBac 6XHis Tra2 PC.14
The plasmid pftz-Luc was constructed as described for
pM-Luc, except that sequences from the ftz gene including its
intron were inserted instead of those from Tra2. The plasmids
pftz-ISS-Luc, pftz-mhc-Luc, and pftz-ISS-mhc-Luc are all
modifications of the pftz-Luc reporter. The sequences used in
ftz-ISS were amplified from pftz12014 and ligated into pftz-Luc
by replacing an ApaI and XhoI fragment. The plasmid pftzmhc-Luc was made using through three PCR amplifications
with the mhc 3' splice site sequence integrated into two different primers:
ftz RNA: Sense-ATGGACTACT TGGACGTCTA CTCG
mhc 3' 1: Antisense-CTTGTTTGCA AGGGGATAAG
TTCAATGGGT TAGCTAATGA GTTTT;
mhc 3' 2: Antisense-GTCTGACGGG TGCGTTTCGA
GTCTTTGCAA TCTTGTTTGC AAGGGGATAA G;
mhc 3' 3: Antisense-CTCGAGCTCC AGGGTCTGGT
AGCGGGTGTA CGTCTGACGG GTGCGTTTCG A.
The plasmid pftz-ISS-mhc-Luc was derived from sequences of
pftz-mhc-Luc in a similar way. Only first step primer is different:
1404
Figure 8. Hfp isoforms differ in nuclear/cytoplasmic localization. (A)
Endogenous Hfp localization (Endo Hfp) detected with a monoclonal antibody directed at the protein’s C-terminal region is shown in
comparison to the localization of Flag-Hfp58 and Flag-Hfp68 in cells
transfected with constructs expressing each form. Anti-Flag antibody
was used to stain transfected Hfp protein. Both Hfp (green) and DAPI
immunfluoescent staining (blue) are overlaid on DIC images of the
same cells in the bottom row. (B) The percentage of signal detected
within the nucleus as determined by Imaris, is shown for both FlagHfp58 and Flag-Hfp68. The number of analyzed cells is indicated as N.
Error bars represent SD values.
ISS-mhc 3' 1: 3-CTTGTTTGCA AGGGGATAAG
TTCAAAAATA AGATTATCTT GCGGTTCG.
The plasmid pFlag-Hfp68 was generated using the same vector as pT2, which has the actin 5C promoter and SV40 PA signal sequence. Flag tagged Hfp68 sequences were amplified from
a cDNA (AT08368) purchased from the Drosophila Genome
Resource Center using primers with integrated Flag sequences.
pFlag-Hfp58 was generated by a similar strategy just with a different start position.
Cell culture and transfection. Dmel/S2 cells were cultured in
SFII-900 medium at 28 degrees and split at regular intervals. For
transfections, 2.5 × 105 cells were seeded in 1.9 cm2 wells and a
total of 1.5 μg of DNA was mixed with 3 μL cellfectin in 100 μL
RNA Biology
Volume 10 Issue 8
of the same medium. A plasmid with no insert that contained
both the Actin5C promoter and SV40 polyA signal was used as
a control and to equalize DNA amounts in all transfections. For
the RNAi screen, 1.5 × 106 cells were seeded in 9.5 cm2 wells and
transfected with 5 μg DNA and 10 μL cellfectin. After 5–6 h,
the cells were resuspended and counted. They were then reseeded
into 96-well plates with 3 × 104 cells per well.
Synthesis of dsRNA. Double-stranded RNAs were synthesized following the approach of Park and Graveley.48 Briefly,
cDNA fragments amplified and inserted into PCR vectors
(Invitrogen). M13 reverse and M13 forward primers were used to
amplify linear DNA template. These fragments were transcribed
using the Megascript kit (Ambion) with SP6, T3, and T7 RNA
polymerases to produce single strand RNAs. Single strand RNAs
were mixed in annealing buffer (100 mM NaCl, 20 mM TRISHCl, pH 8.0, 1 mM EDTA), incubated at 80 degrees for 10 min
and then cooled at room temperature for 30 min, followed by
storage on ice. Primer sequences used to amplify fragments for
dsRNA synthesis are provided in Table S1.
RNA interference. In all secondary RNA interference experiments dsRNAs was added directly to cells growing in SFII-900
media in the amounts of 4 μg per well in 24-well plates, 20 μg
per well in 6-well pate. To maximize the extent the knockdown
on target expression, the RNAi treatment was repeated at both
24 and 48 h after the initial treatment.
RNA immunoprecipitation. For RNA immunopreciptation
(RIP) experiments, 1.0 × 107 cells were seeded in a 78 cm2 dish
and transfected with a total of 30 μg plasmid DNA. After 48 h,
the cells were washed two times with 1X PBS buffer. They were
dounced repeatedly in RIP buffer (150 mM NaCl, 50 mM Tris
pH 7.5, 5 mM EDTA, and 0.05% NP-40). Cell lysates were
pre-absorbed with Sepharose Gammabind beads (Amersham)
for 1 h and then incubated with activated beads with or without conjugated anti-Hfp antibody at 4 °C for 4 h. After washing beads three times, the whole precipitates were treated with
Trizol (Invitrogen). RNA isolated from precipitates was analyzed
by RT-PCR.
Splicing reporter luciferase assays. Luciferase assay were performed following the protocol described in the manual of DualLuciferase reporter assay system kit (Promega). Briefly, 100 μL
passive lysis buffer (Promega) was added to cells in each well of
a 24-well plates. Then 25 μL of the lysate was used to measure
luciferase signals. Luciferase signals were measured on a Perkin
Elmer VICTOR™ X5 Multilabel Plate Reader.
Immunoblotting. Cultured cells were lysed in passive lysis
buffer (Promega). A monoclonal antibody against C terminus of
Hfp30 or the M2 anti-Flag epitope (Sigma) were detected using
appropriate secondary antibodies and the ECL system (General
Electric). After incubating with antibodies for one hour at room
temperature, blots were washed 5 min with TBST (50 mM
TRIS-HCl pH 7.4, 150 mM NaCl, 2.5 mM KCl, 0.05% Tween
20) for three times then exposed to film. Fly samples were
dounced in lysis buffer (20 mM TRIS-HCl pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100), spin at 10 000 rpm for
1 min at 4 degrees. The supernatant was heated in SDS loading
buffer for 10 min.
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Real-time PCR. For quantitative PCR experiments cDNA
was synthesized from 1 μg of total cellular RNA using the
Superscript first strand RNA kit (Invitrogen). The cDNA was
mixed with a 2X Syber Green PCR mix (ABI) and DNA primers
at a final concentration of 0.625 ng/μL. Q-PCR was performed
on 7900 FAST PCR machine (Applied Biosystems) with SDS
2.3 software (Applied Biosystem). Fragments were amplified by
first incubating at 50 °C for 2 min and 95 °C for 10 min, and
then for 40 cycles at 95 °C for 15 sec each and 60 °C for 1 min.
Relative quantification analysis was performed by using the delta
delta CT method.49
S2 cell immunostaining and image analysis. Localization of
proteins was determined after S2 cells were seeded into 12-well plate
with 5 × 105 cells/well. Coated coverslips (BD Biocoat) were placed
into the wells and cells were allowed to settle on them overnight.
Plasmids were transfected the next day. After 36–48 h incubation
at 28 °C, cell medium was removed. Cells were washed once with
1XPBS, then fixed with 4% paraformaldehyde in PBS at °C for
30 min. After washing with PBS, the cells were incubated in PBX
(0.2% triton X-100 in PBS) at room temperature for 10–15 min.
Blocking was performed with 1% BSA at room temperature for
1 h, cells were then incubated with primary antibody (1:200 in
PBS with 1% BSA) at 4 °C overnight. They were then washed with
PBS four times and incubated in secondary antibody buffer (1:500
in PBS with 1% BSA) at room temperature for 1 h. Coverslips were
removed and samples were mounted with CellMask (Invitrogen).
Images were obtained using a Nikon Eclipse Ti Confocal microscope. Images were analyzed for nucleus/cytoplasm distribution using Imaris 7.3 software (Bitplane) after deconvolution by
AutoQuant X3 (MediaCybernetics). The total protein signal
across the entire three dimensional nucleus was divided by that
across the whole cell.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Shihuang Su for the assistance in preparing media
and supplies, Rong Dong for support with Drosophila cultures
and media. We also thank Dr Clifford Stephen in the William S
Dunn Chemical Genomics Screening Center for his help arraying dsRNA used in the RNAi screen. We are grateful to Trudi
Schupbach for providing hfp mutant flies, Hank Adams for
assistance with confocal microscopy, Dr Marco Marcelli and
Huiying Sun for providing facilities of luciferase measurement
and quantitative PCR. This work was supported by an NIH
grant R01GM070892 and a supplement from the American
Recovery and Reinvestment Act (WM) and NIH grant
R00GM080447 and R01 CA167752 (EJW). DNA sequencing was performed at the U.T. MD Anderson DNA Analysis
Facility and supported by a grant (no. CA16672, DAF) from
the National Cancer Institute.
Supplemental Materials
Supplemental materials may be found here:
www.landesbioscience.com/journals/rnabiology/article/25645
RNA Biology
1405
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