Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.
Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.
Holen
Torgeir
T
The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway.
Amarzguioui
Mohammed
M
Wiiger
Merete T
MT
Babaie
Eshrat
E
Prydz
Hans
H
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D020868:Gene Silencing; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D013925:Thromboplastin