Figure 3.

Identification of the Htra2-β1-responsive element within SMN exon 7. (A) SMN1 minigenes containing mutations within SE1 or SE3 showed an increased level of SMN1Δ7 in the absence of Htra2-β1 (lanes 1 and 7). Cotransfection of Htra2-β1 revealed an 1.7-fold increase of FL expression to the SE1 mutant template (lanes 3) and a 2.9-fold increase FL expression to the SE3 mutant (lane 9). Disruption of the AG-rich element, SE2, resulted only in SMN1Δ7 expression (lane 4) and could not be overcome even at the highest concentration of Htra2-β1 (lane 6). (B) Combined disruption of SE1 and SE3 resulted in exclusive SMN1Δ7 expression in the absence of cotransfected Htra2-β1 (lane 2) and could be only slightly compensated by cotransfection of 3 μg Htra2-β1 (lane 4). The SE2 mutation with either SE3 (lanes 5–7) or SE1 (lanes 8–10) mutations resulted exclusively in SMN1Δ7, indicating that SE2 is necessary for the inclusion of exon 7 and the site through which Htra2-β1 functions. (C) Smaller disruptions dividing SE2 into three subdomains (SE2a, SE2b, and SE2c) resulted in low levels of SMN1Δ7 from minigenes with SE2a (lane 1) and SE2b (lane 4) mutations. Transient expression of 3 μg Htra2-β1 resulted in a partly reversion of SMN1Δ7 in FL-SMN1 shown by a 50% decrease of SMN1Δ7 transcript (lanes 3 and 6). The disrupted SE2c minigene resulted in the exclusive expression of SMN1Δ7 (lane 7) and also could not be compensated by transient expression of Htra2-β1 (lanes 8 and 9).