Purification of the pre-spliceosomal E complex formed on hF1γ exon 9–10 pre-mRNA in vitro. (A) The schematic representation of pre-mRNAs, CDC14-15, ftz, hF1γ3GCAUG and hF1γΔGCAUG, fused with two MS2-binding sites at the 3′ exons. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). Average and standard deviation (SD) of the ratio of U1 snRNA to U2 snRNA from three independent experiments are shown at the bottom. (C) Western blotting of the purified E complexes on the pre-mRNAs using U1-70K and U2AF65 antibodies (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (D) The sequences of 5′ splice sites of four kinds of pre-mRNAs as shown in (A). Capital and lowercase letters correspond to exonic and intronic residues, respectively. The nucleotide different from the consensus site is shown in bold.

Splicing of hF1γ exon 9–10 pre-mRNA in U1- or U2-disrupted Xenopus oocytes. (A) Northern blotting of endogenous U1 and U2 snRNAs in Xenopus oocytes. Antisense oligonucleotide against U1 or U2 snRNA was injected into the cytoplasm of Xenopus oocytes to disrupt endogenous U1 or U2. The same volume of water was injected as a control (–). Note that U1 snRNA was truncated by the injection of the U1 antisense oligo (the fast migrating band indicated as U1*), since the oligo targets the 5′ end of U1 snRNA. (B) Splicing of the ³²P-labeled CDC14-15, ftz and hF1γ 9–10 pre-mRNAs in the nuclei of control (lanes 2, 6 and 10), U1-disrupted (lanes 3, 7 and 11), or U2-disrupted oocytes (lanes 4, 8 and 12). The hF1γ 9–10 transcript lacks the intron 8 sequence. RNA was analyzed immediately (lanes 1, 5 and 9) or at 70 min (lanes 2–4, 6–8 and 10–12) after injection.

U1-independent splicing is indispensable for the regulation of alternative splicing of hF1γ by Fox-1. (A) The schematic representation of wild-type (WT) and mutant (mtU1) hF1γ mini-genes (left) and the suppressor U1 snRNP (right). The mutations introduced into the 5′ splice site of exon 9 are underlined. Closed circles indicate the Fox-1 binding element. (B) Transfection assay of hF1γ WT and 5′SSmt mini-genes co-expressed with pCS2+MT vector or pCS+MT-Fox-1 in HeLa cells. The left panel shows spliced products amplified by the oligonucleotides annealed with exons 8 and 10. The middle and right panels show the splicing reaction between exons 8 and 9 and between exons 9 and 10. Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed four times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (C) Suppressor U1 snRNA experiments in HeLa cells. The wild-type or mutant U1 snRNA was co-expressed with wild-type hF1γ mini-gene and pCS2+MT vector or pCS+MT-Fox-1. The spliced products were analyzed by RT-PCR using the oligonucleotides annealed with exons 8 and 10 (left panel), exons 8 and 9 (middle panel) and exons 9 and 10 (right panel). Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed three times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (D) Western blotting of cell extracts using the anti-Myc antibody (upper panel) and anti-U2AF65 antibody as a loading control (lower panel). Molecular size markers are shown on the right.

hF1γ exon 9–10 pre-mRNA is spliced efficiently in U1-inactivated HeLa nuclear extracts in vitro. Splicing reaction of AdML (lanes 1–4), CDC14-15 (lanes 5–8), ftz (lanes 9–12) and hF1γ 9–10 (lanes 13–16) with the normal HeLa cell nuclear extracts (lanes 2, 6, 10 and 14), the U1- (lanes 3, 7, 11 and 15) or U2-inactivated (lanes 4, 8, 12 and 16) extracts, as well as transcripts without incubation (lanes 1, 5, 9 and 13). Pre-mRNA, intermediates and splicing products are indicated schematically on the right.

Role of the 5′ splice site sequence in U1-independent splicing. (A) Base-substitutions introduced into the 5′ splice site of hF1γ3GCAUG pre-mRNA are shown (–3G > C and +5a > g). Capital and lowercase letters correspond to exonic and intronic nucleotides, respectively. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (C) Splicing reaction of hF1γ3GCAUG (lanes 1–3) and hF1γmt5′SS (lanes 4–6). These pre-mRNAs were incubated under the standard in vitro splicing condition for indicated time above each lane. Pre-mRNA, intermediates and splicing products are indicated schematically on the right.