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. 2006 Dec;26(23):8791-802.
doi: 10.1128/MCB.01677-06. Epub 2006 Sep 25.

Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F

J Barrett Crawford  1 , James G Patton
Affiliations

Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F

J Barrett Crawford et al. Mol Cell Biol. 2006 Dec.

Abstract

The inclusion of exons 2 and 3 of alpha-tropomyosin is governed through tissue-specific alternative splicing. These exons are mutually exclusive, with exon 2 included in smooth muscle cells and exon 3 included in nearly all other cell types. Several cis-acting sequences contribute to this splicing decision: the branchpoints and pyrimidine tracts upstream of both exons, UGC-repeat elements flanking exon 3, and a series of purine-rich enhancers in exon 2. Previous work showed that proteins rich in serine-arginine (SR) dipeptides act through the exon 2 enhancers, but the specific proteins responsible for such activation remained unknown. Here we show that a 35-kDa member of the SR protein family, 9G8, can activate the splicing of alpha-tropomyosin exon 2. Using RNA affinity chromatography and cross-linking competition assays, we also demonstrate that the heterogeneous nuclear ribonucleoproteins (hnRNPs) H and F bind to and compete for the same elements. Overexpression of hnRNPs H and F blocked 9G8-mediated splicing both in vivo and in vitro, and small interfering RNA-directed depletion of H and F led to an increase in exon 2 splicing. These data suggest that the activation of exon 2 is dependent on the antagonistic activities of 9G8 and hnRNPs H and F.

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Figures

FIG. 1.
FIG. 1.
9G8 activates α-TM exon 2 inclusion. (A) Exons 1 to 4 of α-TM and splicing regulatory elements are shown, encompassing the branchpoint/pyrimidine tracts of exons 2 (B2P2) and 3 (B3P3), upstream and downstream regulatory elements (URE and DRE), and purine-rich enhancers in exon 2 (denoted by vertical lines). A portion of the exon 2 sequence is shown below, with the four enhancers underlined, denoted B, A, X, and M (20). Previous work showed that only the two central enhancers are needed for activity (5, 20). (B) A minigene (400 ng) encompassing the first four exons of α-TM (pSVpA α-TM 1-4) were cotransfected with 800 ng of eight individual SR protein expression vectors. Transfections were carried out in PAC1 cells, and RT-PCR products were analyzed by dideoxy termination primer extension. The graph below shows averages and standard errors derived from at least three independent transfections. (C) Mutation of the A and X elements (AAAAGAGAAG to AAAACTTAAG, GGAGGAC to GGAGCTT) abolished activation by 9G8. Samples were analyzed and quantitated as described above.
FIG. 2.
FIG. 2.
9G8 activates in vitro splicing of a heterologous construct containing the exon 2 enhancers. (A) In vitro splicing reactions were performed with the indicated substrates in either 40% HeLa nuclear extract, 40% cytoplasmic S100 extracts or 40% S100 extract supplemented with purified SR proteins. The two central exon 2 enhancers elements were inserted into an enhancerless doublesex construct (dsx-ΔE) to make wild-type dsx-TM, whereas enhancer mutations identical to those in Fig. 1 were used to create dsx-AX. After splicing, radiolabeled RNAs were separated on 8% gels with the identity of individual bands as indicated. (B) Individual recombinant SR proteins were added to S100 splicing reactions, and spliced products were analyzed as in panel A. Reactions included increasing amounts of SRp20, ASF/SF2, 9G8, and SRp55 (250 nM, 750 nM, and 1.5 μM). The lanes utilizing substrates with mutated enhancer elements (AX) used 1.5 μM concentrations of each SR protein.
FIG. 3.
FIG. 3.
9G8, hnRNP H, and hnRNP F bind to the exon 2 enhancer elements. (A) RNA oligonucleotides containing wild-type or mutant exon 2 enhancers were covalently linked to modified Sepharose. HeLa nuclear extracts were passed over the columns, and associated proteins were eluted with increasing salt, with a final elution in urea. Fractions were separated on 10% SDS gels, and Western blots performed with antibodies to 9G8. (B) Fractions from both columns were silver stained. The indicated bands were excised and identified by mass spectrometry.
FIG. 4.
FIG. 4.
hnRNP H and hnRNP F antagonize 9G8-mediated activation. (A) In vitro splicing reactions were performed with the enhancer-containing dsx-TM in the presence of 1.5 μM 9G8 and increasing amounts of hnRNPs H and F (750 nM, 1.5 μM, and 3 μM). Similar amounts of PTB and SRp20 were added as controls. (B) The wild-type α-TM construct (pSVpA α-TM 1-4; 400 ng) was cotransfected into PAC1 cells with increasing amounts of hnRNP H or hnRNP F (100 ng, 800 ng, and 3.2 μg), and splicing patterns were analyzed as in Fig. 1. At the highest transfection levels, hnRNP H and hnRNP F fractions were increased 3.1- and 3.4-fold, respectively (see Fig. S2 in the supplemental material). (C) Western blots of whole-cell lysates from PAC1 and HeLa cells were performed with antibodies to hnRNP H, hnRNP F, 9G8, and α-tubulin.
FIG. 5.
FIG. 5.
hnRNPs H and F directly compete with 9G8 for binding to the exon 2 enhancers. (A) Radiolabeled wild-type α-TM transcripts (25 nM) were incubated with 1 μM 9G8 in the presence or absence of increasing amounts of hnRNPs H and F. Reactions were subjected to UV cross-linking, and labeled proteins were analyzed on 10% SDS gels. At least three independent competitions were performed, and averages and standard errors are shown at right (hnRNP H, ▪; hnRNP F, formula image). (B) RNA sequences used for cross-linking (TM, AX, A, X, and INSIDE; panels A and C) and in vitro splicing (M1, M2, and M3; panel D) assays. The wild-type sequence consists of the two central exon 2 enhancers (bracketed above) and the sequence between them (shown in Fig. 1A). Nucleotides underlined and in boldface denote mutated nucleotides. (C) UV cross-linking competition. Radiolabeled wild-type TM RNAs (25 nM) were incubated with 9G8 (▪), hnRNP H (□), or hnRNP F (formula image) in the presence of increasing amounts of cold competitor RNAs. Samples were subjected to UV cross-linking and analyzed as described above. Competitor RNAs were incubated at the indicated molar excess to radiolabeled RNA. RNAs are as indicated in panel B (TM, AX, A, X, and INSIDE). At least three independent competitions were performed, and averages and standard errors are as shown. (D) In vitro splicing reactions were performed with the enhancer-containing dsx-TM and the dsx-M1, M2, M3, and AX mutants in the presence of 9G8 alone (1.5 μM) or when combined with hnRNPs H or F (3 μM).
FIG. 6.
FIG. 6.
siRNA depletion of hnRNP H and hnRNP F. (A) Wild-type pSVpA α-TM 1-4 minigene (400 ng) was cotransfected into PAC1 cells with 10 nM concentrations of siRNAs directed against hnRNP H and/or hnRNP F, either alone, together, or in combination with 800 ng of 9G8. Splicing patterns were analyzed as described above. (B) At least three independent transfections as in panel A were performed, and averages and standard errors for exon 2 inclusion are shown. (C) Western blots of normal cells (mock) or cells treated with siRNAs against hnRNPs H and F were performed with antibodies to hnRNP H, F, or α-tubulin as a loading control.
FIG. 7.
FIG. 7.
siRNA depletion of 9G8. (A) Wild-type pSVpA α-TM 1-4 minigene (400 ng) was cotransfected into HeLa cells with 20 nM concentrations of each of four individual siRNAs (si-1, si-2. si-3, and si-4) directed against 9G8. Western blots were performed on normal cells (mock) or cells treated with siRNAs against 9G8 using antibodies to 9G8 or α-tubulin. (B) Splicing patterns were analyzed as in Fig. 1, and the average and standard errors from at least three independent transfections are shown below (C). (D) Wild-type pSVpA α-TM 1-4 minigene (400 ng) was transfected into HeLa cells with either 20 nM of the si-4 siRNA directed against 9G8 or the si-4 siRNA plus cotransfection of an epitope-tagged (T7) version of 9G8. Western blots were performed on normal cells (mock) or cells treated with siRNAs against 9G8 using antibodies to 9G8, Τ7, or α-tubulin. (E) Splicing patterns were analyzed as in Fig. 1, and the average and standard errors from at least three independent transfections are shown at bottom (F).
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