Download Analysis of alternative splicing in Drosophila genetic

Title: Analysis of alternative splicing in Drosophila genetic mosaics
Shihuang Su1, Diana O’Day1,2, Shanzhi Wang1,2 and William Mattox1,2*
1Department
of Genetics, University of Texas, M.D. Anderson Cancer Center, Houston, TX; 2Genes
and Development Graduate Program, University of Texas, Graduate School of Biomedical Sciences,
Houston, TX
*Address correspondence to William Mattox, Department of Genetics, Unit 1010, University of Texas,
M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009, fax: 713-834-6339; Email: [email protected]
1. Abstract
Many studies on alternative pre-mRNA splicing are carried out in cell free systems or in
cultured cells grown under non-physiological conditions. The introduction of splicing
reporter genes into the genomes of model organisms offers a way to investigate the
functions of splicing regulators in the context of living organisms. This permits the
direct study of alternative splicing events that change during development, aging and in
response to environmental conditions. In these systems powerful genetic tools can be
employed to analyze splicing factor function including conditional expression, targeted
mutations and RNA interference. We present one approach that utilizes Drosophila
genetic mosaics in combination with splicing reporters to visualize how alterations in
splicing factor function affect splice site selection in vivo.
Keywords: Drosophila, development, genetic mosaic, transgenic
2. Theoretical Background
2.1 Reporter genes for splicing in living organisms
As our understanding of the biochemical activities of splicing regulators increases there is a
growing need to understand the behavior of these factors in the context of both normal and
diseased tissues. The introduction of splicing reporter genes into the genome of model
organisms provides a way to visualize the activities of splicing factors in whole organisms and
has several unique advantages. Most importantly, splicing regulation can be explored in a
natural physiological context and simultaneous observations can be made on a variety of
tissues. In cases where splicing is developmentally regulated, the ability to monitor splicing
over time in differentiating tissues has the potential to reveal information about the dynamics
of splicing factor function. Finally, model systems offer a variety of genetic tools that make it
possible to manipulate the activity of splicing regulators and thus test their functions. As the
analysis of splicing in vitro often involves non-physiological reaction conditions, protein
concentrations and post-translational modifications, a genetic approach provides an important
way in which the biological significance of proposed splicing factor functions can be evaluated.
2.2 The introduction of splicing reporters into the Drosophila genome
Strains expressing splicing reporter genes can be generated in a variety of ways. In Drosophila
transgenic strains are usually produced using the phiC31 integrase to introduce a plasmid
carrying an attB site at any of several established genomic attP insertion sites (Bischof et al.,
2007; Groth et al., 2004). Alternatively “knock-in” strategies can be employed to precisely
integrate reporter protein sequences into the genome via homologous recombination at
locations near an alternative splice site of interest (Rong and Golic, 2000).
2.3 The application of genetic mosaics to analysis of alternative pre-mRNA splicing.
A common issue in the analysis of reporters in whole organisms is that the effects of different
genotypes must be inferred from comparison of reporter signals in tissues derived from
separate individuals. Such comparisons are potentially subject variations in the age, sample
preparation, culture conditions and genetic background of individuals within the culture.
These variables can make it difficult to consistently measure changes in splicing ratios. One
solution to this issue is provided by Drosophila genetic technologies that allow the generation
of clones of cells with altered regulator levels in a field of normal cells. Using such genetic
mosaics even subtle changes in the activity of a splicing reporter can be visualized by direct
comparison of cells growing side by side within the same tissue of the same individual. In
addition, because such clones typically encompass only a small fraction of the cells in the
organism, they seldom impair survival. Thus even splicing factors with essential roles in
viability can be studied.
A variety of strategies for mutant clone production have been developed but one of the
most convenient is the “flip-out” strategy (Basler and Struhl, 1994; Ito et al., 1997), which is
diagrammed in the Overview Figure. This method makes use of an inducible transgene
encoding the FLP site-specific recombinase (Golic and Lindquist, 1989) to cause synthesis of
the yeast GAL4 transcription activator by excision of an FRT flanked cassette that otherwise
blocks GAL4 expression (Ito et al., 1997). When FLP expression is induced early in
development, numerous isolated clones of cells expressing GAL4 are produced in all tissues of
the organism. These clones are marked using a second reporter gene under the control of
several UAS elements that are responsive to GAL4. Finally the strategy includes the splicing
reporter transgene and a UAS driven construct that alters the activity of a splicing factor of
interest (cDNA, RNAi, etc). Although the requirement for five different transgenes may seem
onerous, strains bearing most of these components in convenient combinations are available
from public Drosophila stock collections and are widely used within the research community.
3. Protocol
3.1 Transgenes used in flip-out studies
P{hsFLP}: Encodes the yeast FLP site specific recombinase under the control of the inducible
heat shock protein 70b promoter (Golic and Lindquist, 1989).
P{AyGAL4}: Expresses yeast GAL4 from the ubiquitous Actin5C gene promoter after a yellow+
flanked by FRT sites is excised by FLP recombinase (Ito et al., 1997).
P{UAS-eGFP}: Several transgenes are available that express eGFP in response to GAL4 and
can be used to mark the location of clones. For compatibility with GFP-based splicing
reporters a variety of alternative UAS transgenes are available expressing the E. coli lacZ gene
and other markers. If antibodies are available for the splicing factor of interest, this marker
transgene is not necessary.
UAS-dsRNA transgenes: Express dsRNA in response to GAL4 causing knockdown of the
targeted gene’s expression within FLP induced clones. Transgenic strains targeting a wide
variety of splicing factors and other genes are available from the Bloomington Drosophila
Stock Center, Vienna Drosophila Resource Center (Austria) and the National Institute of
Genetics (Japan).
UAS-cDNA transgenes: To overexpress a splicing factor of interest in GAL4 clones a cDNA
may be inserted into a UAS vector such as pAttB-UAST (Bischof et al., 2007) and used to
produce transgenic strains. In the example shown in Figure 1 we used a cDNA from the
Drosophila transformer-2 gene (Qi et al., 2006).
P{EP} insertions: These strains contain random inserts of P transposons bearing UAS
elements and a basal promoter near their 3’ terminus (Rorth, 1996). In cases where the
insertion is near or within the 5’ UTR of a gene of interest overexpression is induced by GAL4
in clones. A variety of strains are available from public collections.
Splicing reporter genes: Splicing reporters, in which the expression of lacZ, an epitope tag, or
various fluorescent proteins (RFP, YFP, GFP) depend on splicing, are amenable to this
procedure provided that a compatible marker protein construct is used to detect GAL4 clones.
The reporter gene should be expressed from either its native promoter or one that generates
fairly uniform staining across the tissue of interest (i.e. Actin5C or tubulin) so that levels inside
and outside the clone can be compared.
3.2 Generation of mosaic flies
Mosaic flies are generated by crossing strain homozygous for hsFLP, AyGAL4 and the UASeGFP marker to one carrying both the splicing reporter and a UAS driven transgene that
affects the expression of a splicing factor of interest as illustrated in the example shown in
Figure 1. Typically, eight male and eight virgin female flies are placed together in a vial with
fly media for 24 hrs to allow mating to occur. They are then transferred to a fresh vial and the
first vial is discarded. The adult flies are allowed to deposit eggs at 25oC in the second vial for
12 hours, they are then removed from this vial and the embryos are develop for an additional 24
hours at 25oC. The vials are then subjected to heat shock to induce expression of the FLP
recombinase. The heat shock is carried out in a covered water bath at 37oC for 30 minutes to
ensure rapid and complete warming of the media. After heat shock incubate the vial for three
more days at 25oC until the larvae have reached their late third instar stage in which they are
noticeably larger (about 2-3 mm in length) and some are beginning to crawl up the walls of the
vial. Larvae are then removed from the vial and dissected as described below. This timeline
works well for obtaining large clones in imaginal discs, but it is important to note that the
timing of FLP induction should be selected based on the properties of the tissue in which clones
will be observed. In all cases the heat shock should occur before cell division has been
completed. If desired, a series of egg lays can be set up and heat shocks can be performed at
several different developmental time points to identify the optimal period for clone generation.
3.3 Immunostaining of Drosophila tissues
In cases where two compatible fluorescent protein reporters are used (i.e. eGFP to mark clones
and Red-Stinger for the splicing reporter gene) it is usually possible to observe the position of
clones and the effects on the splicing reporter by fluorescence microscopy without antibody
staining. However, when enhanced signal is required of a non-fluorescent reporter protein is
used, immunoflourescent staining is carried out.
The desired tissue is dissected under a stereomicroscope using No. 5 forceps (Dumont)
from several 3rd instar larvae immersed in PBT (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
2 mM KH2PO4, 0.1% Triton X100) and immediately place them into 500µl of a solution of 4%
paraformaldehyde in PBT for 30 minutes at room temperature. While observing the dissected
tissues under the microscope the fixative solution is carefully removed using a pipette. The
sample is then washed repeatedly in PBT. To block non-specific antibody binding the tissue is
then placed in 500 µl PBT supplemented with 5% Normal Goat Serum (Sigma) at room
temperature and incubated for 1hour. The tissue is then immersed in PBT + 5% NGS along
with primary antibodies directed at both the protein produced by the splicing reporter (in this
example beta-galactosidase) and the protein used to mark the position of flip-out clones (GFP).
Antibody concentrations and incubation conditions should be optimized depending on the
commercial preparations used. With commercial rabbit beta-galactosidase and mouse GFP
antibodies (Invitrogen) an overnight incubation at 4oC is typically performed using a 1:300
dilution of both antibodies. The tissues are again washed extensively with PBT to remove the
excess primary antibody and then incubated for 2 hours at room temperature with secondary
antibodies (i.e. a 1:100 dilution of goat anti-mouse IgG conjugated to Alexa fluor 488 nM and
goat anti-rabbit IgG Alexa fluor 546 nM from Invitrogen). A variety of other secondary
antibodies may be used, however it is important that each secondary antibody is non-cross
reactive with other primary antibodies present in the staining reactions. In cases where the
staining pattern of the two antibodies is expected to be coincident it is advisable to carry out
separate control staining reactions in which individual primary antibodies are omitted. Finally
the tissues are washed with PBT and carefully transferred to a microscope slide where they are
mounted in 80 µl of Vectashield medium (Vector Laboratories). A 22 mm cover slip is applied
and then sealed with nail polish.
Observe and record staining using an appropriate fluorescent or confocal microscope.
4. Example of an experiment
In Figure 1 we show results from a flip-out experiment in which a UAS-tra2 cDNA is induced
in clones of imaginal disc tissue. The expression of this transgene induces beta-galactosidase
activity from a ubiquitously active tra2 splicing reporter (Mattox and Baker, 1991) in a
coincident pattern indicating that Tra2 autoregulates splicing of its own pre-mRNA in the disc.
5. Troubleshooting
Problem
Flip out clones not detected
with GFP antibody. No live
GFP fluorescence found.
Flip out clones not detected
with GFP antibody but live
GFP fluorescence is detected.
Splicing reporter is unaffected
in clones.
Reason
FLP was induced after cell
division is complete in the
target tissue.
Solution
Carry out heat shock at
several earlier stages of
development.
Alteration in splicing factor
expression causes lethality of
GFP-positive cells.
Non-optimal conditions for
antibody staining
Use a different transgene to
alter splicing factor levels.
Targeted factor acts through
a non-cell-autonomous
pathway to affect splicing
Optimize antibody
concentration and incubation
period or
use live GFP as marker.
Use a non-clonal approach
6. Figure Legends
Figure 1. Induction of splicing reporter by the Transformer-2 (Tra2) protein. Shown is an
imaginal disc in which a large clone of cells expressing the Drosophila Tra2 protein from a
UAS-cDNA transgene was generated using the flip-out system. A lacZ splicing reporter,
transcribed by a promoter active throughout the disc, is designed to detect the amount of
retention of an intron known to be regulated by Tra2 (Mattox and Baker, 1991; Qi et al., 2007).
A: The position of the clone is marked by expression of eGFP from a UAS-eGFP transgene. B:
Elevated expression of LacZ in the clone indicates that intron retention is increased in the
presence of elevated Tra2.
7. References
Basler, K., and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb
pattern by hedgehog protein. Nature 368, 208-214.
Bischof, J., Maeda, R.K., Hediger, M., Karch, F., and Basler, K. (2007). An optimized
transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad
Sci U S A 104, 3312-3317.
Golic, K.G., and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific
recombination in the Drosophila genome. Cell 59, 499-509.
Groth, A.C., Fish, M., Nusse, R., and Calos, M.P. (2004). Construction of transgenic Drosophila
by using the site-specific integrase from phage phiC31. Genetics 166, 1775-1782.
Ito, K., Awano, W., Suzuki, K., Hiromi, Y., and Yamamoto, D. (1997). The Drosophila
mushroom body is a quadruple structure of clonal units each of which contains a virtually
identical set of neurones and glial cells. Development 124, 761-771.
Mattox, W., and Baker, B.S. (1991). Autoregulation of the splicing of transcripts from the
transformer-2 gene of Drosophila. Genes Dev 5, 786-796.
Qi, J., Su, S., and Mattox, W. (2007). The doublesex splicing enhancer components Tra2 and
Rbp1 also repress splicing through an intronic silencer. Mol Cell Biol 27, 699-708.
Qi, J., Su, S., McGuffin, M.E., and Mattox, W. (2006). Concentration dependent selection of
targets by an SR splicing regulator results in tissue-specific RNA processing. Nucleic Acids
Res 34, 6256-6263.
Rong, Y.S., and Golic, K.G. (2000). Gene targeting by homologous recombination in
Drosophila. Science 288, 2013-2018.
Rorth, P. (1996). A modular misexpression screen in Drosophila detecting tissue-specific
phenotypes. Proc Natl Acad Sci U S A 93, 12418-12422.
8. Abbreviations
attB = attachment site B
attP = attachment site P
GFP = Green fluorescent protein
IgG = Immunoglobulin G
NGS = Normal Goat Serum
tra2 = transformer2