Regulation of Retention of FosB Intron 4 by PTB

One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed ΔFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3′ end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4

PTB and U2AF⁶⁵ bind to the 3′ end of fosB I4.

<p>(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.</p

<i> In vivo</i> splicing of fosB I4 is regulated by PTB.

<p>(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 <i>fosB</i> 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.</p

Oligodeoxynucleotides used in subcloning, PCR, UV cross-linking and mutagenesis studies.

<p>Oligodeoxynucleotides used in subcloning, PCR, UV cross-linking and mutagenesis studies.</p

PTB binds to the 3′ end of fosB I4.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000828#pone-0000828-g001" target="_blank">Fig. 1</a>. The position of protein molecular weight markers is indicated on the right in kDa.</p

Alkaline phosphatase treatment results in the lack of PTB binding to the 3′ end of fosB I4.

<p>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.</p

Comparison of fosB I4 sequence among vertebrates.

<p>(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 <i>fos b</i> 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.</p