Figure 7.
Figure 7. Rbfox3 represses exon 12 of Numb via its direct binding to the UGCAUG elements in intron 11. (A) Requirement for endogenous Rbfox3 for exclusion of exon 12 in Numb mRNAs. The indicated siRNAs were electroporated with the GFP construct in the neural tube at E2. GFP+ cells were isolated by FACS from E4 embryos (see Fig. 3 B). The Numb mRNAs from GFP+ cells were analyzed by RT-PCR. Because the data in Fig. 3 C and panel A of this figure were from the same experiment, the “Actin” panels in these two figures are the same. (B) Enhancement of Numb exon 12 exclusion by forced expression of Rbfox3-full. The Rbfox3-full-ires-GFP construct or the GFP construct was electroporated in the neural tube at E2. GFP+ cells were isolated by FACS from E4 embryos. The Numb mRNAs from GFP+ cells were analyzed by RT-PCR. (C) In vivo binding of Rbfox3 to Numb transcripts. Top: a diagram of the chicken Numb premRNA surrounding alternative exon 12. Exon 11 and exon 13 are constitutive exons. Rectangles and horizontal lines indicate exons and introns, respectively. Two copies of the UGCAUG element in intron 11 are shown as ovals. Bottom: detection of intron 11 of the Numb transcript associated with Rbfox3. E4 embryo extracts were subject to RNA–protein coimmunoprecipitation using the indicated antibodies. The intron 11 region of the Numb transcripts associated with the immunocomplex was analyzed by PCR (38 cycles) using primers P9 and P10 after incubation with reverse transcriptase (RT, +) or without RT (−). RT-PCR (33 cycles) of Numb mRNAs using primers P5 and P6 (see Fig. 6 A) serves as a negative control that lacks the UGCAUG element. (D) Direct binding of Rbfox3 to the UGCAUG elements of Numb intron 11 RNA in vitro. Top: a diagram of the upstream intronic silencers (UISs) in Numb intron 11 RNA, which includes wild-type or mutant UGCAUG elements. Bottom: after UV cross-linking of Rbfox3 with the indicated radiolabeled RNA probes, Rbfox3 was immunoprecipitated and subjected to SDS-PAGE. Autoradiography of SDS-PAGE (myc-Rbfox3) and input RNA probes after Urea-PAGE (Input) are shown. (E) A diagram of Numb minigene. The minigene includes the chicken Numb genomic DNA fragment containing exons E11 and E12 and intron 11, which are flanked by exons E2 and E3 of the rat preproinsulin gene (PPI). Transcription of the minigene is driven by the Rous sarcoma virus long terminal repeat (RSVLTR). Four different minigenes that contain the four different UISs, identical to the UISs shown in D (top), were constructed. (F) UGCAUG-dependent repression of exon 12 by Rbfox3. The minigenes containing the indicated UISs were cotransfected into SK-N-SH cells with the Rbfox3 expression construct and exon 12 splicing patterns of the minigene mRNAs were analyzed by RT-PCR using primers P11 and P12 (E).

Rbfox3 represses exon 12 of Numb via its direct binding to the UGCAUG elements in intron 11. (A) Requirement for endogenous Rbfox3 for exclusion of exon 12 in Numb mRNAs. The indicated siRNAs were electroporated with the GFP construct in the neural tube at E2. GFP+ cells were isolated by FACS from E4 embryos (see Fig. 3 B). The Numb mRNAs from GFP+ cells were analyzed by RT-PCR. Because the data in Fig. 3 C and panel A of this figure were from the same experiment, the “Actin” panels in these two figures are the same. (B) Enhancement of Numb exon 12 exclusion by forced expression of Rbfox3-full. The Rbfox3-full-ires-GFP construct or the GFP construct was electroporated in the neural tube at E2. GFP+ cells were isolated by FACS from E4 embryos. The Numb mRNAs from GFP+ cells were analyzed by RT-PCR. (C) In vivo binding of Rbfox3 to Numb transcripts. Top: a diagram of the chicken Numb premRNA surrounding alternative exon 12. Exon 11 and exon 13 are constitutive exons. Rectangles and horizontal lines indicate exons and introns, respectively. Two copies of the UGCAUG element in intron 11 are shown as ovals. Bottom: detection of intron 11 of the Numb transcript associated with Rbfox3. E4 embryo extracts were subject to RNA–protein coimmunoprecipitation using the indicated antibodies. The intron 11 region of the Numb transcripts associated with the immunocomplex was analyzed by PCR (38 cycles) using primers P9 and P10 after incubation with reverse transcriptase (RT, +) or without RT (−). RT-PCR (33 cycles) of Numb mRNAs using primers P5 and P6 (see Fig. 6 A) serves as a negative control that lacks the UGCAUG element. (D) Direct binding of Rbfox3 to the UGCAUG elements of Numb intron 11 RNA in vitro. Top: a diagram of the upstream intronic silencers (UISs) in Numb intron 11 RNA, which includes wild-type or mutant UGCAUG elements. Bottom: after UV cross-linking of Rbfox3 with the indicated radiolabeled RNA probes, Rbfox3 was immunoprecipitated and subjected to SDS-PAGE. Autoradiography of SDS-PAGE (myc-Rbfox3) and input RNA probes after Urea-PAGE (Input) are shown. (E) A diagram of Numb minigene. The minigene includes the chicken Numb genomic DNA fragment containing exons E11 and E12 and intron 11, which are flanked by exons E2 and E3 of the rat preproinsulin gene (PPI). Transcription of the minigene is driven by the Rous sarcoma virus long terminal repeat (RSVLTR). Four different minigenes that contain the four different UISs, identical to the UISs shown in D (top), were constructed. (F) UGCAUG-dependent repression of exon 12 by Rbfox3. The minigenes containing the indicated UISs were cotransfected into SK-N-SH cells with the Rbfox3 expression construct and exon 12 splicing patterns of the minigene mRNAs were analyzed by RT-PCR using primers P11 and P12 (E).

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