Dynamic Antagonism between ETR-3 and PTB Regulates Cell Type-Specific Alternative Splicing  Nicolas Charlet-B, Gopal Singh, Thomas A. Cooper  Molecular Cell  Volume 9, Issue 3, Pages 649-658 (March 2002) DOI: 10.1016/S1097-2765(02)00479-3 Copyright © 2002 Cell Press Terms and Conditions

Figure 1 ETR-3 Binds to U/G Dinucleotide Motifs in cTNT MSE2 and MSE3 (A) The MSE1-4 RNA contains four muscle-specific splicing elements. (B) UV crosslinking binding assays contained 0.6 μg recombinant GST-tagged ETR-3 and 20 μg HeLa nuclear extracts with the indicated uniformly labeled RNA. The stronger signal for MSE2 compared to MSE3 is probably due to more labeled uridines near the binding sites. Competition and filter binding assays demonstrate equivalent binding (data not shown). (C) Scanning mutagenesis identifies specific ETR-3 binding sites as U/G motifs (red) in MSE2 and MSE3. Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 2 ETR-3 Regulates MSE-Dependent Splicing In Vitro (A) cTNT splicing substrate. (B) Time course of 32P-labeled RNA product and intermediate accumulation during in vitro splicing in HeLa nuclear extract displayed on an 8% denaturing gel. (C) Primer extension of in vitro splicing reactions using 5′ end-labeled oligo complementary to exon 6. (D) Primer extension analysis of in vitro splicing reactions containing 0.2 or 0.8 μg of purified recombinant His-ETR-3. The elution buffer for recombinant protein had no effect on splicing (data not shown). The same results were obtained with two different preparations of recombinant ETR-3 (data not shown). Representative results from four independent trials are shown. Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 3 A Dominant-Negative CELF Mutant Blocks MSE-Dependent Activation of Exon Inclusion in Embryonic Skeletal Muscle (A) Domain structure of full-length and truncated CELF4. (B) Structure of the cTNT minigene. (C) CELFΔ blocks ETR-3-mediated activation of exon inclusion in fibroblasts. An ETR-3 expression vector (0.4 μg) was cotransfected with 0.3 and 1.0 μg of CELFΔ expression vector and 0.1 μg of the cTNT minigene. RT-PCR results are representative of three transfections. Lower panel: Western blot analysis of tagged CELFΔ and ETR-3 proteins using AntiXpress antibody. The RT-PCR and Western blot analyses shown were performed on plates transfected in parallel. (D) CELFΔ inhibits MSE-dependent exon inclusion in embryonic skeletal muscle cultures. The CELFΔ expression plasmid (0.3 μg) was transfected where indicated (+). Representative gels are shown. Percent exon inclusion is the average with standard deviations from at least three independent transfections. Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 4 PTB Binds to MSEs 1, 2, and 4 (A) Immunoprecipitation of PTB UV crosslinked to MSE1-4 RNA in HeLa nuclear extracts. Pellet, P; Supernatant, S. (B) Binding of endogenous PTB in HeLa nuclear extracts to MSE1-4 was challenged with individual unlabeled MSE RNAs (2, 6, 18 pmol). (C) Scanning mutagenesis to map PTB binding sites in MSEs 1, 2, and 4. Binding of either endogenous PTB in HeLa nuclear extracts (MSE1 and MSE2) or purified recombinant PTB (MSE4) was assayed. Comparable results were obtained using endogenous and recombinant PTB on MSEs 2 and 4 (data not shown). Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 5 MSE-Dependent Repression of cTNT Exon Inclusion by PTB In Vivo and In Vitro (A) Coexpression of the PTB expression vector (0.3 μg) with MSE-containing or MSE-lacking minigenes in muscle cultures. Splicing of minigene mRNA was assayed by RT-PCR. (B) Primer extension analysis of E46NB in vitro splicing with addition of 2.5, 5.0, or 10 pmol unlabeled MSE4 RNA (lanes 2–4) or MSE4mut RNA (+97 +119, Figure 4C) (lanes 8–10). Lanes 5–7 contain 10 pmol unlabeled MSE4 RNA competitor and 0.2, 0.4, or 0.8 μg PTB. (C) Primer extension analysis following addition of 0.2 and 0.4 μg of purified recombinant His-ETR-3 (lanes 3 and 4). Lanes 5–8 contain 0.4 μg of His-ETR-3 and 0.2, 0.4, 0.6, and 0.8 μg of purified recombinant His-PTB, respectively. The largest amount of recombinant His-PTB elution buffer used (lane 8) did not affect in vitro splicing (data not shown). (D) Recombinant His-PTB (0.25, 0.5, 1, 2 μg) blocks binding of recombinant His-ETR-3 (50 ng) to MSE2. UV crosslinking was performed in the presence of 3 μg yeast tRNA and 3 μg BSA. Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 6 A PTB Dominant-Negative Mutant Derepresses Exon Inclusion in Nonmuscle Cells (A) Domain structure of full-length PTB and the PTBΔ dominant-negative mutant. (B) PTBΔ expressed in vivo does not bind MSE1-4. Untransfected HeLa (−), HeLa cultures transfected with full-length PTB (PTB), or dominant-negative PTB (PTBΔ). Supernatants (S) (one-tenth of sample) and pellets (P) (one-third of sample) from immunoprecipitation were analyzed by SDS-PAGE and then either dried down and exposed to film to detect protein-32P-RNA adducts or transferred to PVDF membrane for Western blot analysis using AntiXpress antibodies. The asterisk marks the position of the Ig heavy chain. (C) Coexpression of PTBΔ with cTNT minigenes results in MSE-dependent derepression of exon inclusion in nonmuscle cells. QT35 fibroblasts were transfected with an MSE-containing or MSE-lacking minigene with 0.3, 1.0, or 3.0 μg PTBΔ expression vector. Equivalent expression of PTBΔ was demonstrated by Western blots (data not shown). Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions

Figure 7 Dominant-Negative Mutants for CELF and PTB Demonstrate Both Activities in Both Muscle and Nonmuscle Cells (A) Coexpression of an MSE-containing minigene with PTB and CELF dominant-negative mutants in QT35 fibroblasts. Transfections included 1.0 μg PTBΔ and 0.3 or 1.0 μg CELFΔ expression vectors. Lower panel: Western blot results using antibodies against the AntiXpress epitope tag on both PTBΔ and CELFΔ demonstrating that PTBΔ expression is not affected by CELFΔ coexpression. (B) Coexpression of an MSE-containing minigene with PTB and CELF dominant-negative mutants in primary skeletal muscle cultures (0.3 μg of PTBΔ and CELFΔ expression plasmids). Western blot (lower panel) demonstrates that CELFΔ is not decreased by coexpression of PTBΔ. The RT-PCR and Western blot analyses shown were performed on plates transfected in parallel. Molecular Cell 2002 9, 649-658DOI: (10.1016/S1097-2765(02)00479-3) Copyright © 2002 Cell Press Terms and Conditions