RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.
In posttranscriptional gene silencing (PTGS), "quelling," and RNA interference (RNAi), 21-25 nucleotide RNA fragments are produced from the initiating dsRNA. These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).
Lipardi
C
C
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Wei
Q
Q
Paterson
B M
BM
D000818:Animals; D004330:Drosophila / Q000196:embryology; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016372:RNA, Antisense; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000096:biosynthesis* / Q000378:metabolism
D008164:Luminescent Proteins; C034477:RNA primers; D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D049452:Green Fluorescent Proteins; D012313:RNA; D008836:Micrococcal Nuclease