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

被引:40
作者
Chu, JX [1 ]
Dolnick, BJ [1 ]
机构
[1] Roswell Pk Canc Inst, Dept Pharmacol & Therapeut, Buffalo, NY 14263 USA
来源
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE | 2002年 / 1587卷 / 2-3期
关键词
chemotherapy; thymidylate synthase; RNA editing; antisense RNA;
D O I
10.1016/S0925-4439(02)00081-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The 3' untranslated region (UTR) of rTSalpha 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 rTSalpha RNA and TS mRNA, suggesting a possible effect of rTSalpha RNA on TS mRNA levels. HEp2 cells expressing a Tet-On transactivator were transiently co-transfected with pHook-1 and a construct containing rTSalpha (protein and antisense RNA), rTSalpha-3' (rTSalpha protein only), rTSalpha-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 rTSalpha or rTSalpha-3 but not rTSalpha-3' or luciferase, resulted in decreased TS mRNA levels as measured by RPA. These results demonstrated that the antisense region of rTSalpha 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 rTSalpha or rTSalpha-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. (C) 2002 Elsevier Science B.V All rights reserved.
引用
收藏
页码:183 / 193
页数:11
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