ROLE OF RNASE-H IN HYBRID-ARRESTED TRANSLATION BY ANTISENSE OLIGONUCLEOTIDES
ROLE OF RNASE-H IN HYBRID-ARRESTED TRANSLATION BY ANTISENSE OLIGONUCLEOTIDES
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DOI：
10.1073/pnas.85.14.5011
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发表时间：
1988-07-01
影响因子：
11.1
通讯作者：
WALDER, JA
中科院分区：
文献类型：
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作者：
WALDER, RY;WALDER, JA
已经用兔网织红细胞裂解物系统研究了反义寡脱氧核苷酸杂交阻止翻译的机制。研究的寡核苷酸针对小鼠α-或β-珠蛋白mRNA的不同区域。发现新鲜制备的网织红细胞裂解物含有有核细胞中 1-2% 的 RNase H 水平。在主动翻译的条件下，这种活性水平足以在 1 小时内在与互补寡脱氧核苷酸杂交的位点切割近 100% 的靶 mRNA。使用聚[rA)oligo(dT)作为酶的竞争性抑制剂，发现与跨越起始密码子的序列或编码区中的序列互补的寡脱氧核苷酸的杂交停滞完全是由于RNase H对mRNA的切割。与β-珠蛋白mRNA而非α-珠蛋白mRNA的帽位点相邻的寡脱氧核苷酸的杂交也直接抑制蛋白质合成。然而，即使在这种情况下，RNase H 对 mRNA 的切割也是主要的抑制途径。
The mechanism of hybrid-arrested translation by antisense oligodeoxynucleotides has been investigated with the rabbit reticulocyte lysate system. The oligonucleotides studied were directed against different regions of mouse .alpha.- or .beta.-globin mRNAs. Freshly prepared reticulocyte lysates were found to contain 1-2% of the level of RNase H in nucleated cells. This level of activity was sufficient to cleave nearly 100% of the targeted mRNA at the site of hybridization with a complementary oligodeoxynucleotide in 1 hr under conditions of active translation. Using poly[rA)oligo(dT) as a competitive inhibitor of the enzyme, hybrid arrest by oligodeoxynucleotides complementary to the sequence spanning the initiation codon or to a sequence in the coding region was found to be due entirely to cleavage of mRNA by RNase H. Hybridization of oligodeoxynucleotides adjacent to the cap site of .beta.-globin mRNA, but not the .alpha.-globin mRNA, also inhibited protein synthesis directly. Even in this case, however, cleavage of the mRNA by RNase H was the predominant pathway of inhibition.