FIG. 2.
XIAP IRES RNP core binding sites maps within 28-nt segment of the IRES element. (A) Nucleotide sequence of the 103-nt portion of XIAP IRES (probe C). The positions of oligonucleotides used for binding site mapping are shown under the sequence. The approximate position of the RNP core binding site is indicated by a bracket above the sequence. The polypyrimidine tract is shown in a shaded box. The AUG codons are underlined. The point mutation at position −27 (G to U) which introduces an out-of-frame AUG, is indicated above the sequence. (B) Oligonucleotide mapping of the XIAP IRES RNP core binding site. Indicated oligonucleotides were annealed to the 103-nt [32P]RNA probe (probe C) and the gel mobility shift assays were performed using S100 extracts from HeLa cells. The position of the XIAP IRES RNP complex is indicated. (C) Deletion and mutational analysis of XIAP IRES element. DNA segments corresponding to the indicated regions of the XIAP 5′ UTR were inserted into the XhoI site of the linker region of the plasmid pβgal/CAT. The small solid boxes indicate the position of the polypyrimidine tract. The brackets represent the position of the RNP core binding site. HeLa cells were cotransfected with the indicated plasmids as described in Materials and Methods. The levels of βGal and CAT activity were determined at 48 h posttransfection. Relative CAT activity was calculated by normalizing to βGal activity. Expression of each CAT cistron from the pβgal/hUTR/CAT construct was set at 100%. The bars represent the average ± SD of three independent transfections.