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Figure 1 Conservation of 4.1R E16 and flanking intron sequences. Conserved sequence elements are boxed. M, mouse; h, human; f, frog; c, chicken; b, bovine; introns are in lower case and exons in upper case.
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 | Figure 2 Mutation of CE16 activates E16 inclusion. (A) 4.1R minigene and E16 wild-type sequence with 4.1/PRE16-dsx, and linker-scan sequence substitutions (underlined) in E16 aligned below. All mutant constructs are the same length as the wild-type minigene. (B) RT−PCR assay of spliced products from wild-type 4.1R minigene and the PRE16-dsx substitution mutation. In vivo splicing in transfected HeLa cells (lanes 1−3), in HeLa nuclear extract (lanes 4 and 5) and in Xenopus oocytes (lanes 6 and 7) with the E16 inclusion/exclusion ratio. (C) RT−PCR results of in vitro splicing of CE16 linker-scanning mutations of the 4.1R minigene in HeLa nuclear extract (lanes 8−15).
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Figure 3 4.1R E16 sequence elements were tested for exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) activities in a heterologous pre-mRNA context. (A) Schematic diagram of the dsx constructs with the position of primers used for RT−PCR indicated by arrows. (B) RT−PCR assay of splicing for each of the dsx constructs. Splicing was performed in microinjected oocytes.
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 | Figure 4 4.1R pre-mRNA splicing in the presence of excess CE16 RNA competitor (A) or in HeLa cell nuclear extract depleted with CE16 RNA (B). (A) Splicing was performed in the absence of competitor (lanes 1 and 6) or in the presence of increasing amounts of CE16 RNA competitor (lanes 2−5) or control RNA (lanes 7−10). Amounts of competitor added are indicated above each lane (pmol), and the E16 inclusion/exclusion ratio is shown below each lane. (B) Splicing was performed in complete nuclear extract (lane 11), in nuclear extract depleted by pre-incubation with 10 or 20 pmol of CE16 RNA (lanes 12 and 13), or in extract mock depleted with control RNA (lane 14).
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Figure 5 CE16 RNA affinity isolation of candidate silencer protein(s). (A) Coomassie Blue stain of proteins eluted from CE16 RNA after incubation in HeLa nuclear extract. Bands of  33−35 kDa are hnRNP A/B proteins (lane 1) as identified by nanospray mass spectrometry. SA, streptavidin from magnetic beads. Lane 2 shows molecular weight standards. (B) Western blot analysis of HeLa nuclear extract (lanes 3 and 6) and of proteins eluted from CE16 and control RNAs performed with antibody against hnRNP A1/A1B (lanes 3−5) and hnRNP A2/B1 proteins (lanes 6−8). (C) Western blot of proteins eluted from biotinylated CE16 and silencer mutant RNAs performed with antibodies against hnRNP A1 and A2.
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 | Figure 6 Inhibition of E16 splicing by recombinant hnRNP A/B proteins. (A) In vitro splicing assay. Recombinant hnRNP A1 protein silences E16 splicing in CE16-depleted extract (lanes 1−3) and in complete extract (lanes 4−6). (B) In vivo splicing assay. HeLa cells were transfected with 4.1R minigene alone (lane 8) or co-transfected with 4.1R plus either pCG-hnRNP Al expression plasmid (lane 9) or the empty pCG expression vector (lane 10). Lane 7 is a mock transfection. (C) In vitro splicing assays with the indicated recombinant hnRNP A1 variants. Splicing was performed in HeLa nuclear extract in the absence of added A1 (lanes 11 and 15), or in the presence of wild-type A1 (lanes 12 and 16), A1 with mutant RRMs (lanes 13 and 17) or truncated UP1 variant (lanes 14 and 18). (D) In vitro splicing assays in the absence of added A1 (lanes 19 and 24) or with the indicated recombinant hnRNP A/B protein isoforms (lanes 20−23 and 25−28).
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Figure 7 Temporal relationship between 4.1R E16 splicing and the expression of hnRNP A/B proteins in differentiating mouse erythroblasts. (A) RT−PCR analysis of E16 splicing patterns in erythroblasts cultured for 0−44 h in the presence of erythropoietin (lanes 1−4). (B) Western blot analysis of expression of 4.1R + E16 and splicing factors in erythroblasts cultured for 0−44 h (lanes 5−8). Protein 4.1R isoforms including the peptide encoded by E16 were detected using anti-peptide antibody 10-1; splicing factors were assayed using antibodies against hnRNP A1, A2/B1, hnRNP H and hnRNP I/PTB.
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