Munroe S H
Cold Spring Harbor Laboratory, NY 11724.
EMBO J. 1988 Aug;7(8):2523-32. doi: 10.1002/j.1460-2075.1988.tb03100.x.
Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.
与人类β-珠蛋白前体mRNA或嵌合珠蛋白/腺病毒E2a前体mRNA互补的反义RNA在体外能特异性且高效地抑制前体mRNA剪接。抑制水平取决于反义RNA相对于前体mRNA底物的长度、位置和浓度。与珠蛋白3'剪接位点下游大于80个核苷酸的序列互补的反义RNA的抑制效率至少与跨越剪接位点的反义RNA相同。因此，剪接对涉及距剪接位点有一定距离的外显子序列的扰动敏感。抑制作用由影响反义RNA与底物RNA退火的因素介导。对RNA双链体形成的直接分析表明，在HeLa细胞核提取物中存在一种活性，它以不依赖ATP的方式促进互补RNA的快速退火。当在底物加入后大于或等于5分钟时将反义RNA添加到剪接反应中，退火和抑制作用都会大大降低。这一结果可能反映了一种开放结构（其中反义RNA与前体mRNA的退火更容易）和一种封闭复合物（其中前体mRNA在剪接体组装的早期阶段被隔离）之间的转变。
