Chu Jianxiong, Dolnick Bruce J
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Biochim Biophys Acta. 2002 Jul 18;1587(2-3):183-93. doi: 10.1016/s0925-4439(02)00081-9.
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), rTSalphaDelta3' (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 rTSalphaDelta3' 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.
rTSα RNA的3'非翻译区（UTR）与人类胸苷酸合成酶（TS）RNA互补（即反义）。当HEp2细胞（人表皮样癌）从对数生长期后期进入平台期生长时，核糖核酸酶保护试验（RPA）显示rTSα RNA水平与TS mRNA水平呈负相关，提示rTSα RNA可能对TS mRNA水平有影响。表达Tet-On反式激活因子的HEp2细胞与pHook-1以及包含rTSα（蛋白质和反义RNA）、rTSαDelta3'（仅rTSα蛋白质）、rTSα-3'（反义RNA-荧光素酶）或荧光素酶的构建体进行瞬时共转染。对转染细胞进行筛选，并评估诱导的转基因表达对TS mRNA的影响。通过RPA检测，转染的rTSα或rTSα-3'（而非rTSαDelta3'或荧光素酶）的诱导表达导致TS mRNA水平降低。这些结果表明，rTSα RNA的反义区域对于TS mRNA的这种下调是必要且充分的。对TS mRNA的RPA还显示，在rTSα或rTSα-3'转染的细胞中出现了两个部分长度的受保护片段，且其出现频率增加。RPA严谨性评估和引物延伸试验表明，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中发挥作用。
