RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs

Cell. 2001 Nov 2;107(3):297-307. doi: 10.1016/s0092-8674(01)00537-2.

Abstract

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).

MeSH terms

  • Animals
  • Drosophila / embryology
  • Gene Silencing*
  • Green Fluorescent Proteins
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Micrococcal Nuclease / metabolism
  • Polymerase Chain Reaction
  • RNA / metabolism*
  • RNA Processing, Post-Transcriptional*
  • RNA, Antisense
  • RNA, Double-Stranded / metabolism*
  • RNA, Messenger / metabolism*
  • RNA, Small Interfering
  • RNA, Untranslated / biosynthesis*
  • RNA, Untranslated / metabolism

Substances

  • Luminescent Proteins
  • RNA primers
  • RNA, Antisense
  • RNA, Double-Stranded
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA, Untranslated
  • Green Fluorescent Proteins
  • RNA
  • Micrococcal Nuclease