Intronic Alus Influence Alternative Splicing
Figure 4
Distance effect of Alu elements on the alternative splicing pattern.
(A) A schematic illustration of the genomic region between exons 2 and 3 of RABL5 gene. Arrows marked A and B indicate two positions where an intronic sequence was inserted. (B) An 800-nucleotide intronic sequence was inserted in site B. The 800-nucleotide sequence was gradually shortened to the size shown above each lane. The indicated wt and chimeric plasmids were transfected into human 293T cells, total RNA was collected and examined by RT-PCR analysis (lanes 1–9). Lanes 10–12 show insertions of a different sequence, containing 25 nucleotides without any known splicing regulatory sequences, into the same site. This sequence was duplicated and triplicated to generate 50- and 75-nucleotide inserts. These mutant RABL5 minigenes were examined as above. (C) Similar analysis as in panel B, except that the 800-nucleotide sequence and the shorter sequences were inserted into site A. Spliced products are shown on the right and each PCR product was confirmed by sequencing. Splicing products were separated on a 2% agarose gel. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software.