Figure 2.

Splicing activity of the cassette tolerates many, but not all, mutations to the bordering codons. (A) Schematic representation of the TFIIIA-eGFP gene construct showing the location of the mutated bordering codons (labeled NNN). The eGFP sequence is shown in green. Aberrant spliced products (red lines) are described in the text and the sequences are shown in Supplementary Figure S6. (B, C) Comparison of spliced products detected by RT–PCR for select 5′-ss and 3′-ss mutations tested within the TFIIIA-eGFP context (also see Supplementary Figure S2). Mutants were coexpressed with AtL5 (+) or LUC as a control (−). Spliced products are labeled and the sequences are shown in Supplementary Figure S6. The mutants are categorized as fully functional (green), semi-functional (yellow) or non-functional (red). Red nucleotides indicate the mutations made to the bordering codon, and the corresponding encoded amino acid is listed below. (D) Fold induction of protein expression quantified by eGFP fluorescence for select TFIIIA-eGFP mutants (also see Supplementary Figure S2). Induction with AtL5 was measured by comparison to induction with LUC as a control. For each mutant construct, the two amino acids encoded by the 5′- and 3′-bordering codons are indicated.