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首页> 外文期刊>Biochimica et biophysica acta. Molecular basis of disease: BBA >Natural antisense (rTSα) RNA induces site-specific cleavage of thymidylate synthase mRNA
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Natural antisense (rTSα) RNA induces site-specific cleavage of thymidylate synthase mRNA

机译：天然反义（rTSα）RNA诱导胸腺嘧啶合酶mRNA的位点特异性切割

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The 3' untranslated region (UTR) of rTSα RNA is complementary (i.e., antisense) to human thymidylate synthase (TS) RNA. When HEp2 cells (human epidermoid carcinoma) progressed from late-log to plateau phase growth, ribonuclease protection assay (RPA) revealed an inverse correlation between the levels of rTSα RNA and TS mRNA, suggesting a possible effect of rTSα RNA on TS mRNA levels. HEp2 cells expressing a Tet-On transactivator were transiently co-transfected with pHook-1 and a construct containing rTSα (protein and antisense RNA), rTSα△3' (rTSα protein only), rTSα-3' (antisense RNA-luciferase) or luciferase. Transfected cells were selected and evaluated for the effects of induced transgene expression on TS mRNA. induced expression of transfected rTSα or rTSα-3', but not rTSα△3' or luciferase, resulted in decreased TS mRNA levels as measured by RPA. These results demonstrated that the antisense region of rTSα RNA is necessary and sufficient for this down-regulation of TS mRNA. RPA for TS mRNA also showed the enhanced appearance of two partial-length protected fragments in rTSα or rTSα-3' transfected cells. RPA stringency evaluations and primer extension assays indicated that TS mRNA is cleaved in vivo in a site-specific manner. These data demonstrate that rTS gene expression likely plays a role in down-regulating TS through a natural RNA-based antisense mechanism.
机译：rTSαRNA的3'非翻译区（UTR）与人胸苷酸合酶（TS）RNA互补（即反义）。当HEp2细胞（人类表皮样癌）从晚期对数生长期发展到高原期时，核糖核酸酶保护试验（RPA）显示rTSαRNA和TS mRNA的水平呈负相关，这表明rTSαRNA对TS mRNA的水平可能产生影响。将表达Tet-On反式激活子的HEp2细胞与pHook-1和含有rTSα（蛋白和反义RNA），rTSα△3'（仅rTSα蛋白），rTSα-3'（反义RNA荧光素酶）或萤光素酶。选择转染的细胞并评估诱导的转基因表达对TS mRNA的影响。 RPA可以检测到，诱导转染的rTSα或rTSα-3'（而不是rTSα△3'或荧光素酶）的表达导致TS mRNA水平降低。这些结果证明，rTSαRNA的反义区对于TS mRNA的这种下调是必要和充分的。用于TS mRNA的RPA在rTSα或rTSα-3'转染的细胞中还显示出两个部分长度受保护的片段的增强外观。 RPA严格性评估和引物延伸分析表明，TS mRNA在体内以位点特异性方式被切割。这些数据表明，rTS基因表达可能通过基于天然RNA的反义机制在下调TS中起作用。

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