Development and validation of a minigene system to study the alternative splicing of TAF6.

<p>(A) A schematic diagram showing the workflow used to study <i>cis</i>-acting RNA sequences in TAF6 alternative splicing using a TAF6 minigene plasmid. The plasmid containing an uninterrupted genomic sequence from the <i>taf6</i> gene that includes portions of exons 2 and 3, as well as the natural intron 2 is depicted alone with the positions of primers used to detect exogenously expressed RNA species by RT-PCR. Minigenes were transfected into HeLa and 42 hours later total RNA was isolated for use in RT-PCR with primers from flanking plasmid sequences. PCR products were quantified by analysis using an Agilent Bioanalyzer. The percentage TAF6δ is expressed as a ratio of total spliced TAF6 mRNAs (δ + α). (B) Validation of the minigene system via mutagenesis. The proximal (P) 5′ splice site (SS) and distal (D) 5′ SS are illustrated. Mutations that knock-out (ko) SS or strengthen by creating consensus (cons) SS and their impact on the percentage of TAF6δ produced (x-axis), are indicated. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).