Natural antisense (rTSalpha) RNA induces site-specific cleavage of thymidylate synthase mRNA.
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.
Chu
Jianxiong
J
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Dolnick
Bruce J
BJ
D020413:3' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics; D017393:RNA Editing; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012260:Ribonucleases; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000235:genetics*; D014162:Transfection
D020413:3' Untranslated Regions; D012322:RNA Precursors; D016372:RNA, Antisense; D012333:RNA, Messenger; D008156:Luciferases; D013940:Thymidylate Synthase; D012260:Ribonucleases