FIG. 3.

TIAR binding depends on the base-pairing interaction between U1 snRNA and the pseudo 5′ splice site. (A) A suppressor U1 rescued exon 4 inclusion of the calcitonin/CGRP reporter that contains mutations at the +1 to +3 positions of the pseudo 5′ splice site. RT-PCR assays were carried out with total RNA isolated from HeLa cells transfected with the calcitonin/CGRP reporter with wild-type enhancer (wt; lane 1) or the enhancer that contains a mutated pseudo 5′ splice site (mt; lanes 2 to 4) in the absence (lane 2) or presence (lanes 3 and 4) of U1 plasmids (lane 3, wild-type U1; lane 4, mutant U1 containing the compensatory mutations). The percent inclusion of exon 4 is indicated below each lane, and products are identified as indicated in Fig. 1B. M, molecular weight markers. (B) The suppressor U1 restored TIAR binding to the enhancer that was mutated at the pseudo 5′ splice site. Nuclear extracts were prepared from HeLa cells transfected with wild-type U1 (lanes 1 and 2) or the suppressor U1 (lanes 3 and 4) plasmid and used in the UV cross-linking-IP assays. The UV cross-linking-IP reaction was carried out with the ³²P-labeled RNA containing wild-type (lanes 1 and 4) or mutated (lanes 2 and 3) enhancer sequence. The RNA substrate indicated in Fig. 2A was used in this experiment.