Differential Recruitment of Pre-mRNA Splicing Factors to Alternatively Spliced Transcripts In Vivo
Figure 4
Mutation-Independent Differential Recruitment of Endogenous Pre-RNA Splicing Factors
(A) Stable cell lines expressing minigenes containing a G>A mutation at +3 (tau10+3) or a C>U mutation at +14 (tau10+14). Both mutations give preferential inclusion of exon 10 as previously reported for transient expression.
(B) Quantitative real-time PCR analysis demonstrating the preferential inclusion of exon 10 in tau10+3 and tau10+14 compared to tau10wt. Values represent averages ± standard deviations from at least three experiments. Values of included exon 10 are normalized to total minigene RNA.
(C) Quantitative analysis of recruitment of endogenous splicing factors to tau10+3 or tau10+14. Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using specific probes against the tau minigene and IF microscopy with specific anti-splicing factor antibodies. Percentage of cells with colocalization of tau RNA-FISH and splicing factor signals is indicated. Values represent averages from at least 100 transcription sites from three experiments ± SEM.