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. 2005 Jul;11(7):1084–1094. doi: 10.1261/rna.7263905

FIGURE 5.

FIGURE 5.

Overexpression of La rescues splicing of a splicing deficient mutant in vivo. (A) Reverse transcription reaction analyzing splicing of tdwt and tdSH1 RNA. The DNA primer hybridizes in exon2 and by the addition of dATP, dGTP, dCTP, and ddTTP in the presence of reverse transcriptase either a primer + 5-nt transcript is produced (for pre-mRNA), a primer + 16-nt (for mRNA) or a primer + 8-nt (for cryptic-mRNA). (SJ) Splice junction, (3′ ss) 3′ splice site. (B) Representative autoradiograph of a polyacrylamide gel. Reverse transcription products of mRNA, pre-mRNA, and a cryptic-mRNA are indicated. Cryptic splicing occurs when an alternative fold of the 5′ splice site is formed. The double mutant tdSH1 is significantly reduced in splicing (lane 2). In the presence of La or E. coli protein StpA (lanes 3,5) splicing was significantly increased. In the presence of Ro60 splicing was moderately increased (lane 4). The last lane shows splicing of the wild-type td mRNA. (C) Percentage (mean value ±standard deviation) of splicing [mRNA + cryptic-mRNA] ×100/[mRNA + cryptic-mRNA + pre-mRNA] of three individual experiments.