FIG. 7.

TIAR and Hu proteins regulate binding of U1 and U6 snRNA to the 5′ splice site of NF1 exon 23a. (a) Psoralen cross-linking of the NF1 RNA substrate indicated in Fig. 6a in the presence (lane 2) or absence (lane 1) of HeLa nuclear extract (N.E.). (b) Psoralen cross-linking of the NF1 RNA substrate in HeLa nuclear extract without added protein (lane 1) or with with increasing amounts (0.5 and 2.0 μg) of GST (lane 2 and lane 3), GST-TIAR (lanes 4 to 8), GST-mHuB (lane 9 and lane 10), or a truncated Hu protein, GST-mHuB RRM1,2 (lane 11 and lane 12). A 2′-O′-methyl ribo-oligonucleotide that specifically blocks the pre-mRNA binding region of U1, U2, or U6 snRNA was included to identify the cross-linked species (lanes 6 to 8). (c) Psoralen cross-linking of NF1 RNA substrate in HeLa nuclear extract, in which the endogenous TIA-1 and TIAR proteins were removed by immunodepletion. Lane 1, mock-depleted nuclear extract; lane 2, TIA-1/TIAR-depleted nuclear extract; lane 3, TIA-1/TIAR-depleted nuclear extract supplemented with 1 μg GST-TIAR. The TIA-1/TIAR protein levels in the mock- and TIA-1/TIAR-depleted nuclear extracts were examined by Western blotting. (d) Psoralen cross-linking of NF1 RNA substrate in HeLa nuclear extract (lane 1) and CA77 nuclear extract (lane 2). A Minx RNA substrate that contains a 5′ splice site was used as a control in this experiment (lanes 3 and 4). The levels of TIA-1/TIAR proteins and a U1 snRNP protein were detected by Western blotting. U170K protein was used to indicate the levels of U1 snRNPs in these nuclear extracts. (e) Psoralen cross-liking of the NF1 RNA substrate in HeLa nuclear extract supplemented with increasing amounts (0.5 and 4 μg) of GST-HuR (lane 2 and lane 3), GST-mHuC (lane 4 and lane 5), or GST-HuD (lane 6 and lane 7).