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1.
Figure 6

Figure 6. PTB and U2AF65 bind to the 3′ end of fosB I4.. From: Regulation of Retention of FosB Intron 4 by PTB.

(A) 32P-labeled mini RNA substrate transcribed from the 3′ region of I4 within the ISS/WT construct were incubated in the presence of either control HeLa nuclear extract (lanes 1 and 3) or PTB-depleted HeLa nuclear extract (lanes 2 and 4) before UV cross-linking. The samples were then immunoprecipitated with either an α-PTB antibody (lanes 1 and 2) or an α-U2AF65 antibody (lanes 3 and 4). Samples were resolved by electrophoresis and visualized by autoradiography. (B) 32P-labeled mini RNA substrates transcribed from the 3′ region of I4 within either the ISS/WT (lanes 1 and 3) or ISS/T6 constructs (lanes 2 and 4) were incubated in the presence of HeLa nuclear extract before UV cross-linking. The samples were then immunoprecipitated with either an α-PTB antibody (lanes 1 and 2) or an α-U2AF65 antibody (lanes 3 and 4), resolved by electrophoresis and visualized by autoradiography.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.
2.
Figure 2

Figure 2. PTB binds to the 3′ end of fosB I4.. From: Regulation of Retention of FosB Intron 4 by PTB.

(A) 32P-labeled mini RNA substrates from the 5′, M and 3′ region of I4 were incubated in the absence (−) or presence (+) of HeLa nuclear extract before UV cross-linking, resolved by gel electrophoresis and visualized by autoradiography. (B) 32P-labeled mini RNA substrates from the 5′, M and 3′ region of I4 were incubated in the presence of HeLa nuclear extract before UV cross-linking. The samples were then immunoprecipitated with antibody that recognizes PTB or a control antibody that does not recognize splicing factors, resolved by gel electrophoresis and visualized by autoradiography. The 5′, M and 3′ substrates are transcripts from three regions of the intron as defined in the legends of Fig. 1. The position of protein molecular weight markers is indicated on the right in kDa.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.
3.
Figure 5

Figure 5. Alkaline phosphatase treatment results in the lack of PTB binding to the 3′ end of fosB I4.. From: Regulation of Retention of FosB Intron 4 by PTB.

32P-labeled mini RNA substrates from the 3′ region of fosB I4 were incubated in the presence of purified his-tagged PTB 1 protein and UV cross-linked (lane 1, X). Alternatively, samples were phosphatase treated either after or before the UV cross-linking step (lanes 2 and 3, XP and PX, respectively) or phosphatase digested and kinase treated before or after cross-linking (lanes 4 and 5, PKX and XPK, respectively). Samples were resolved by gel electrophoresis and visualized by autoradiography. The position of a protein molecular weight marker is indicated on the right in kDa.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.
4.
Figure 3

Figure 3. In vivo splicing of fosB I4 is regulated by PTB.. From: Regulation of Retention of FosB Intron 4 by PTB.

(A) HeLa cells were transiently co-transfected with ISS/WT and either a control plasmid (bluescript) and/or a construct containing cDNA corresponding to PTB 1. Addition of the control plasmid ensured equal levels of DNA within the transfection assay. The amounts of PTB 1 plasmid used per well in the transfection assays were 1.5 (dark grey box), 1.0 (stripped box) and 0.5 µg (stippled box). The data is representative of single experiment in which all conditions were performed in duplicate. The splicing products of the fosB mini-gene transcripts were analyzed by semi-quantitative PCR using primers that detect both the intron retained transcript (IR) and the intron spliced transcript (IS). An example of the PCR products generated from ISS/WT transcripts isolated from HeLa cells co-transfected with either bluescript (A) or PTB 1 (B) is shown in the inset. The data is represented as the ratio of IS to IR mRNA. Bars display the standard error, *p<0.05.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.
5.
Figure 1

Figure 1. Comparison of fosB I4 sequence among vertebrates.. From: Regulation of Retention of FosB Intron 4 by PTB.

(A) The filled boxes at the top represent fosB exons. The horizontal lines between the boxes represent the three constitutively spliced introns that are removed from the pre-mRNA generated from the gene encoding FosB. The regulated fourth intron is located between the bold-faced vertical lines. Shown below the pre-mRNA is the I4 sequence from the mouse fos b gene. The sequence to the left of the first//represents the 5′ I4 transcript; the sequence between the two sets of//represents the middle I4 transcript; the sequence to the right of the second//represents the 3′ I4 transcript. The sequence that is underlined is the PTB binding site. (B) Diagrammatic representation of the consensus splice sites, branch point and pyrimidine tract sequences for vertebrates. The human, rat, mouse and fish fosB I4 splice sites are indicated below. Capital letters represent exonic sequence and lowercase letters represent intronic sequence, n denotes any nucleotide, y for pyrimidines, r for purines and p(y)tract for the polypyrimidine tract, the underlined sequence shows the putative decoy 5′ splice site.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.
6.
Figure 4

Figure 4. The CT-rich sequence of fosB I4 is required for PTB binding and fosB transcript splicing in vitro and in vivo.. From: Regulation of Retention of FosB Intron 4 by PTB.

(A) Wild type (ISS/WT) and Mutant (ISS/T6) CT-rich region of fosB I4 sequence. Two nucleotides indicated by asterisks were mutated to disrupt PTB binding (ISS/T6). 32P-labeled mini RNA transcripts generated from the 3′ regions of I4 within the ISS/WT and ISS/T6 constructs were incubated in the presence of increasing amounts of HeLa nuclear extract before UV cross-linking. Samples were then resolved by gel electrophoresis and visualized by autoradiography. (B) In vitro splicing reactions containing 32P-labeled ISS/WT and ISS/T6 fosB pre-mRNA substrate were incubated in HeLa nuclear extract and the resultant products of splicing were resolved by gel electrophoresis and visualized by autoradiography. The size the of RNA markers are shown on the right in nt. IR, IS, IVS-E5 and E4 indicate the intron retained transcript, the intron spliced transcript, the intermediate lariat and exon 4, respectively. (C) RNA products from the gels in (B) were cut out and PCR assays with primers specific for each product were used to amplify the IR, IS and IVS-E5 products. DNA markers are indicated on the right in nt. (D) HeLa cells were transiently co-transfected with ISS/WT (white box) or ISS/T6 mutant fos B mini-gene sequence (dark grey box) and either a control plasmid (bluescript) or a construct containing cDNA corresponding to PTB 1. Addition of the control plasmid ensured equal levels of DNA within the transfection assay. The amounts of PTB 1 plasmid used per well in the transfection assays were 1.5 µg (light grey stippled box), and 0.5 µg (dark grey stippled box). The data is representative of single experiment in which all conditions were performed in duplicate. The splicing products of the fosB mini-gene transcripts were analyzed by semi-quantitative PCR using primers that detect both the intron retained transcript (IR) and the intron spliced transcript (IS). The data is represented as the ratio of IS to IR mRNA. Bars display the standard error, *p<0.01.

Victor Marinescu, et al. PLoS ONE. 2007;2(9):e828.

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