Figure 6. Mutant Rnu2–8 Selectively Decreases Splicing Efficiency of Introns.

(A) The sequences of wild type and deletion (Δ) U2 constructs used for cell transfection experiments.

(B) RPA analysis of RNA from mock-transfected HEK293T cells or cells transfected with wild type (WT) or ΔU2 plasmids.

(C and D) HEK293T cells were transfected with the ISS+ splicing reporter (C) or the ISS− splicing reporter (D), or co-transfected with WT or ΔU2 constructs as indicated. The splicing of pBPLUGA parental vector is shown in (C) as a control, and the splicing of the ISS+ reporter is shown in (D) as a reference. The splicing efficiency of the reporters was measured by luciferase/ beta-gal activity (upper panels; C and D) and confirmed by RT-PCR (lower panels). Values represent mean ± SEM, n = 3; *p<0.01; one-way ANOVA. * and arrows in reporter diagrams denote stop codons and RT-PCR primers, respectively.

(E) A diagram of the 3' region of the L1CAM gene. *, stop codon. Arrows, RT-PCR primers.

(F) RT-PCR was performed on wild type (+/+) and NMF291^−/− (−/−) cerebellar cDNA at indicated ages.

(G and H). HEK293T cells were transfected with L1CAM exon27-intron27-exon28 (G) and exon28-intron28-exon29 (H) splicing reporters with or without wild type or ΔU2 constructs as indicated. Splicing of these reporters was analyzed as described above in C and D. As references, the spliced RT-PCR products of exon27-intron27-exon28 reporter and the unspliced PCR product amplified from the exon28-intron28-exon29 reporter plasmid (DNA) are also shown in (H). Values represent mean ± SEM, n = 3; *p<0.01; one-way ANOVA. * in reporter diagrams, stop codons.