Denman R B, Smedman M, Ju W, Rubenstein R, Potempska A, Miller D L
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Nucleic Acids Res. 1994 Jun 25;22(12):2375-82. doi: 10.1093/nar/22.12.2375.
Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and trans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (beta APP mRNA), 5' decreases GUC decreases 3'. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of beta APP mRNA as well as beta APP pre-mRNA, was shown to cleave a beta APP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of beta APP mRNA were reduced 25-30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of beta APP mRNA were reduced 67-80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state alpha-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of beta APP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of beta APP mRNA in COS-7 cells does not depend on ribozyme cleavage.
构建了两组编码反式作用锤头状核酶和顺式作用发夹状核酶的真核表达载体。在一组载体中，核酶RNA转录受小鼠乳腺肿瘤病毒长末端重复序列（MMTV-LTR）的控制。在另一组中，核酶表达由金属硫蛋白IIA（Mt-IIA）启动子控制。每个核酶都靶向阿尔茨海默病淀粉样肽前体mRNA（β-APP mRNA）中的第一个靶序列，5'端为GUC，3'端为下降序列。从这些载体转录的核酶RNA应能切割β-APP mRNA的所有六种可变剪接形式以及β-APP前体mRNA，已证明其在体外能切割β-APP RNA底物类似物。制备了稳定转染两种载体类型的COS-7细胞系。在存在糖皮质激素的情况下生长的、由MMTV-LTR启动子驱动的含有活性或突变锤头状核酶载体的细胞中，β-APP mRNA的稳态水平降低了25%-30%。在含有由Mt-IIA驱动的核酶的细胞系中，糖皮质激素诱导启动子后，在含有锤头状核酶和发夹状核酶的细胞系中，β-APP mRNA的稳态水平均降低了67%-80%。这些水平与低水平诱导核酶RNA的出现相关。相比之下，这些细胞中α-肌动蛋白mRNA和甘油醛-3-磷酸脱氢酶mRNA的稳态水平保持不变。细胞提取物的蛋白质免疫印迹显示，所有形式的β-APP都相应减少。在仅携带载体的稳定转染对照细胞中未观察到核酶转染细胞系中RNA和蛋白质的减少。这些结果表明，COS-7细胞中核酶介导的β-APP mRNA降解不依赖于核酶切割。
