Natural antisense (rTSalpha) RNA induces site-specific cleavage of thymidylate synthase mRNA.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease(2002)

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摘要
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), rTSαΔ3′ (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 rTSαΔ3′ 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.
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关键词
Chemotherapy,Thymidylate synthase,RNA editing,Antisense RNA
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