Fig 3.
SmB deficiency impairs snRNP assembly and exon 7 splicing. (A) NIH 3T3 cell lines with regulated knockdown of mouse SmB. Western blot analysis of NIH 3T3 wild-type (Control) and NIH 3T3-SmBRNAi cells cultured for 4 days without (−) or with (+) Dox. Blots were probed with antibodies against SmB to monitor knockdown efficiency and tubulin as a loading control. Note that SMN expression is not decreased by SmB deficiency. (B) SmB deficiency impairs snRNP assembly in NIH 3T3 cells. Three increasing amounts of extracts from NIH 3T3-SmBRNAi cells (10 μg, 20 μg, and 40 μg) cultured for 5 days without (−) or with (+) Dox were analyzed by in vitro snRNP assembly with radioactive U1 snRNA. Reaction mixtures were analyzed by gel shift, and known RNA-protein complexes are indicated on the right (top panel) (54). SmB and SMN protein levels in the extracts used for snRNP assembly were analyzed by Western blotting (bottom panels). (C) SmB deficiency decreases the efficiency of exon 7 inclusion in NIH 3T3 cells. Radioactive RT-PCR analysis of exon 7 splicing following transfection of SMN1 or SMN2 minigene reporters into NIH 3T3-SmBRNAi cells cultured without (−) or with (+) Dox. Arrows point to exon 7-included (FL) and exon 7-skipped (Δ7) SMN mRNAs. (D) Quantification of the efficiency of exon 7 inclusion in NIH 3T3-SmBRNAi. Blue and red bars correspond to results from NIH 3T3 cells cultured without (−) or with (+) Dox, respectively. Values represent means and SEM from independent experiments (n=3; **, P < 0.01; ***, P < 0.001) as in panel C. Data were normalized relative to −Dox set as 1.