Fig 1.
SMN deficiency impairs exon 7 splicing. (A) NIH 3T3 cell lines with regulated knockdown of mouse Smn. Western blot analysis of NIH 3T3-SmnRNAi and NIH 3T3-SMN/SmnRNAi cells cultured for 5 days without (−) or with (+) doxycycline (Dox). Blots were probed with an antibody that recognizes both mouse and human SMN proteins to monitor knockdown efficiency, with an anti-Flag antibody to detect epitope-tagged human SMN, and with an antibody against tubulin as a loading control. Addition of Dox to the growth medium specifically induces shRNA expression and consequently RNAi-mediated knockdown of endogenous mouse Smn protein. The slower migration of RNAi-resistant human SMN is due to the presence of an epitope tag at the amino terminus as indicated by its detection with anti-Flag antibodies. (B) Smn deficiency decreases the levels of spliceosomal snRNPs in NIH 3T3 cells. Extracts from NIH 3T3-SmnRNAi and NIH 3T3-SMN/SmnRNAi cells cultured for 5 days either without (−) or with (+) Dox were immunoprecipitated with an anti-Sm antibody followed by 3′ end labeling of RNA, denaturing gel electrophoresis, and autoradiography. Known snRNA species are indicated on the right. Note that most U6 snRNAs harbor a 2′,3′-cyclic phosphate end that is not labeled using this assay. Quantification of snRNP levels from independent experiments is shown in Fig. S2 in the supplemental material. (C) SMN 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-SmnRNAi 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-SmnRNAi. 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=4; *, P < 0.05; ***, P < 0.001) as in panel C. Data were normalized relative to −Dox set as 1. (E) Expression of human SMN corrects exon 7 splicing defects caused by depletion of endogenous mouse Smn in NIH 3T3 cells. Radioactive RT-PCR analysis of SMN exon 7 splicing following transfection with either SMN1 or SMN2 minigene reporters into NIH 3T3-SMN/SmnRNAi cells cultured without (−) or with (+) Dox. (F) Quantification of the efficiency of exon 7 inclusion in NIH 3T3-SMN/SmnRNAi cells. 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) as in panel E. Data were normalized relative to −Dox set as 1.