Effect of Number and Position of CI Cassette Exon Splicing Silencer Motifs

Splicing reporters were constructed with variations in the number and position of UAGG and/or GGGG motifs. Three sets of schematics (boxed at center) illustrate the CI cassette exon and adjacent 5′ splice site region with positions of exonic UAGG (black vertical bars) and 5′ splice site GGGG (grey vertical stripe) motifs. Splicing reporter names are indicated at left. Vertical arrowhead indicates 5′ splice site. Each splicing reporter was generated by site-directed mutagenesis from parent plasmid wt0. Natural UAGG positions 51 and 93 represent the starting position of the motif relative to the first base of the exon. Engineered UAGG positions 11, 76, and 100 are also indicated (see schematic in center box at top). Sequence changes of the mutations are underscored: 11, GUGG→UAGG; 51, UAGG→AUGG; 76, CCAG→UAGG; 93, UAGG→GUGG; 100, UCCAA→UAGGC. Representative splicing patterns in PC12 cells (left gel panels) and C2C12 cells (right gel panels) are shown together with average percent exon inclusion values. The correlation between motif pattern and strength of splicing silencing is summarized (bottom). Exon-included (double arrowheads) and exon-skipped (single arrowheads) products are indicated.