Mutagenesis of G-repeat motifs in HAS1 intron 3 enhances exon 4 skipping.

<p>Selected G-repeat motifs in G345 (striped line) were mutagenized according to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053469#pone-0053469-g003" target="_blank">Figure 3</a>. Splicing profiles driven by various G345 derivatives were analyzed by RT-PCR using E3/E5 primer set and agarose gel electrophoresis (A). Product in box is not FL as determined by DNA fragment analysis (data not shown). Abnormal HAS1 transcripts driven by G345/G1–18 are summarized in (B). PCR products of G345/G1–18 m transfectants were cloned and spliced junctions were identified by sequencing of subclones. Arrows indicate authentic and cryptic donor sites which located 144 and 279 bp downstream of authentic donor site. The strength of each donor site is determined according to splice site prediction by a neural network (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>).