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. 2003 Nov;23(21):7437-47.
doi: 10.1128/MCB.23.21.7437-7447.2003.

Regulation of alternative splicing by SRrp86 and its interacting proteins

Jun Li  1 , Ian C Hawkins, Christopher D Harvey, Jennifer L Jennings, Andrew J Link, James G Patton
Affiliations

Regulation of alternative splicing by SRrp86 and its interacting proteins

Jun Li et al. Mol Cell Biol. 2003 Nov.

Abstract

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

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Figures

FIG. 1.
FIG. 1.
Coimmunoprecipitation of SRrp86-associated proteins. HEK 293T cells were transfected with 10 μg of empty Flag vector or the different Flag-tagged SRrp86 constructs shown in panel A. Cells were harvested after 24 h, and about 850 μg of total cell lysate was immunoprecipitated with anti-Flag M2-conjugated agarose beads and washed in low-salt buffer containing 100 mM KCl. Proteins were eluted in 8 M urea and resolved on 15% SDS gels. (B) Five micrograms of total cell lysate (left side) and one-third of the bound proteins (right side) were resolved on 15% SDS gels and stained with Coomassie blue. Arrows mark the positions of the overexpressed constructs and anti-Flag IgG chains. Note that Flag-SRrp86ΔRS comigrates with the light chain of IgG. The values on the left are the sizes of the markers (lane MW) in kilodaltons. (C) One-third of the eluates was subjected to Western blot analysis with a monoclonal antibody against SRp20. The anti-Flag IgG chains were recognized by the secondary antibody because they are from a mouse.
FIG. 2.
FIG. 2.
In vitro interaction between SRrp86 and SAF-B, hnRNP G, YB-1, p72, or 9G8. Epitope-tagged SRrp86 was created by fusing the TAP tag (47) to the carboxy terminus of SRrp86. This tag consists of a calmodulin binding peptide and two IgG binding sites from protein A separated by a tobacco etch virus protease site. For our purposes, only the IgG binding sites were used. TAP-tagged SRrp86 was purified from baculovirus-infected Hi5 cells and incubated with the indicated 35S-labeled protein. Associated proteins were captured by passage over IgG-conjugated agarose beads. Bound proteins were eluted and analyzed on SDS gels. As controls, in vitro-translated luciferase was also incubated with SRrp86-TAP and background binding to the beads alone was determined for each reaction.
FIG. 3.
FIG. 3.
SRrp86 activates CD44 v5 inclusion in vivo. (A) Schematic diagram of the murine CD44 gene (top) and CD44 v5 minigene (bottom; RSV, Rous sarcoma virus; MCS, multiple cloning site; SV40 poly A, simian virus 40 polyadenylation site). An arrowhead indicates the position of the promoter, and arrows indicate the positions of the PCR primers. (B) Increasing amounts of SRrp86 cDNA were cotransfected with the CD44 v5 minigene into COS7 cells. Spliced products were analyzed by RT-PCR, separated on 6% denaturing gels, and quantified by phosphorimager analysis. The increase in v5 inclusion is graphed below. Exon skipping and inclusion products are diagramed at the right. (C) As in panel B, the effects of increasing amounts of SRrp86ΔEK and SRrp86ΔRS on v5 inclusion were examined in transfected COS7 cells.
FIG. 4.
FIG. 4.
SAF-B, hnRNP G, and 9G8 antagonize SRrp86 in promoting v5 inclusion. (A to D) Increasing amounts of the indicated constructs were cotransfected with the CD44 v5 minigene into COS7 cells, and v5 inclusion was analyzed as described in the legend to Fig. 3. Representative gels are shown, and the increase in v5 inclusion was determined from multiple independent transfection experiments.
FIG. 5.
FIG. 5.
SRrp86 activates the distal 5′ splice site of the E1A minigene. (A) The E1A minigene contains three alternative 5′ splice sites and a common 3′ splice site to generate 9S, 12S, and 13S mRNAs, as shown. (B) Increasing amounts of SRrp86 cDNA were cotransfected with the E1A minigene, and spliced products were analyzed by RT-PCR, separated on 6% denaturing gels, and quantified by phosphorimager analysis. The values on the right indicate the sedimentation values for adenovirus E1a mRNAs. The increase in 9S mRNA and the 12S/13S ratio are shown in panel C.
FIG. 6.
FIG. 6.
9G8 antagonizes SRrp86. (A) HeLa cells were transfected with the E1A minigene and 200 ng of the indicated constructs. Spliced products were analyzed by RT-PCR, separated on 6% denaturing gels, and quantified by phosphorimager analysis. The fold increase in the 12S/13S mRNA ratio is graphed in panel B, and the increase in 9S mRNA is shown in panel C. Averages and standard deviations were determined from multiple independent transfection experiments.
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References

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