Figure 5. Purified FOX-1 protein binds in vitro to multiple sites in intron VI using motifs GCAUG and GCACG.

(A) The diagram of intron VI shows the intron VI fragments (A–E) and smaller regions (RNA oligonucleotides B-45 to E-25) tested for direct FOX-1 binding in vitro. Only RNAs from fragments B, C, and E bind to purified FOX-1 (Figure 5—figure supplement 1C). Motifs GCAUG and GCACG (red) and sequences common to all three fragments (light blue) are shown below the diagram. Blue, yellow, and green rectangles indicate the locations of sequences B-37, C-35, and E-25, respectively, within FOX-1 binding fragments of intron VI. The RNA oligonucleotides listed were used in competition experiments with ³²P-labeled fragment B (panel B) and ³²P-labeled intron VI (panels C and D). (B) Small RNA oligonucleotides corresponding to sequences within fragment B compete for FOX-1 binding in vitro. Graphs show cross-linking competition experiments in which binding of FOX-1 (32 ng) to ³²P-labeled fragment B RNA was challenged with an increasing molar excess of either cold fragment B RNA or small RNA oligonucleotides to sequences in fragment B that are also found in the other FOX-1 binding regions in fragments C and E. Binding is expressed as the percent of ³²P fragment B bound by FOX-1 without any competitor RNA. (C) RNA oligonucleotides compete for FOX-1 binding to intron VI. The cross-linking competition experiments are similar to those in panel (A), except the probe is ³²P-labeled full-length intron VI RNA. Binding is expressed as the percent of ³²P intron VI bound by FOX-1 without any competitor RNA. The finding that the B-15 oligonucleotide fails to compete with either fragment B probe or intron VI probe, while the B-45 and B-37 oligonucleotides compete well, indicates that GCAUG, GCACG, or both are utilized for FOX-1 binding. (D) FOX-1 binds to multiple sites within intron VI using both GCAUG and GCACG. Graphs show results of cross-linking competition experiments in which binding of FOX-1 (32 ng) to ³²P-labeled intron VI RNA was challenged with an increasing molar excess of several cold RNAs, as indicated. Binding is expressed as the percent of ³²P intron VI RNA bound by FOX-1 without any competitor RNA. Cold intron VI RNA carrying deletions of the common sequences in B (Δ37) and C (Δ35) competed very poorly with intron VI probe for FOX-1 binding, and cold intron VI with deletions in all three common regions [B (Δ37), C (Δ35), and E (Δ26)] competed even less efficiently, demonstrating the critical role of these sequences in FOX-1 binding. In contrast, RNA oligonucleotides (C-35, B-37, E-35, and E-25) of sequences in fragments B, C, and E competed very effectively with intron VI for binding to FOX-1, further supporting the conclusion that FOX-1 binds to multiple sites in intron VI. The 25 nt RNA oligonucleotide in fragment E contains only the motif GCACG, but not GCAUG, indicating that GCACG promotes robust FOX-1 binding. The deletion in E (Δ26) is one nucleotide longer than the E-25 oligonucleotide, including deletion of a 3' U. Error bars, SEM.

Figure 5—figure supplement 1. Cross-linking experiments show that FOX-1 binds directly to multiple sites in intron VI.

(A) Graphs show cross-linking experiments comparing binding to ³²P-labeled RNA probes of intact intron VI or intron III with different concentrations of purified FOX-1 protein and different cold RNA carriers to inhibit nonspecific FOX-1 binding. Compared are cold intron III RNA (100-fold molar excess) and cold tRNA (100-fold molar excess). FOX-1 binds directly to intron VI but not intron III. tRNA can partially reduce FOX-1 binding to intron VI. (B) Graphs show cross-linking competition experiments in which FOX-1 binding (32 ng) to ³²P-labeled intron VI RNA was challenged with an increasing molar excess of either cold intron VI or cold intron III RNA. Intron III RNA does not compete with ³²P-labeled intron VI RNA for binding to FOX-1, consistent with it not binding FOX-1 in (A). Binding is expressed as the percent of ³²P intron VI RNA bound by FOX-1 without any competitor RNA. Based on experiments in (A) and (B), cold intron III was chosen as the cold carrier for subsequent cross-linking experiments (C) Multiple subregions within intron VI bind directly to FOX-1. Graphs show the results of cross-linking experiments between different concentrations of purified FOX-1 protein and ³²P-labeled probes of intact intron III, intron VI, and subregions within intron VI. The diagram of intron VI below the graphs shows the subregions tested for direct binding in vitro. These graphs show that only B, C, and E can bind FOX-1, but they do not reveal the relative binding affinities of the different fragments. (D) Graphs show cross-linking competition experiments in which FOX-1 binding (32 ng) to ³²P intron VI RNA was challenged with an increasing molar excess of cold fragment B or cold fragment C RNAs. These graphs confirm that FOX-1 binds to the B and C subregions of intron VI.