RETRACTED: Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class

Abstract

In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex)-cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.

An SNF2 Protein Associated with Nuclear RNA Silencing and the Spread of a Silencing Signal between Cells inArabidopsis

“…In plants, the mechanism is different from that in Caenorhabditis elegans, although, in both organisms, an RNA-dependent RNA polymerase (RDR) is involved Ruby et al, 2006;Pak and Fire, 2007;Sijen et al, 2007). In plants, an RNA becomes a template for RDR if it is targeted by one or two primary siRNAs or by a related type of silencing RNA known as microRNA (miRNA) .…”

Epigenetics: heterochromatin meets RNAi

“…Primary and secondary siRNAs have been demonstrated in C. elegans, having different modifications at the 5′ends [49,50]. Primary siRNAs, which are the products of Dicer activity, have 5′-monophosphates and 3′-hydroxy groups whereas secondary RNAs are de novo synthesized by RDPs and have triphosphates at their 5′ end [49,50]. In plants, as well as the single-cell algae Chlamydomonas reinhardtii, the 3′-end of miRNA/siRNA is methylated and thereby protected from degradation [43,50,51].…”

“…Secondary siRNAs promote the major effects on silencing of mRNAs but also, likely, on transcriptional silencing and heterochromatin formation [48]. Primary and secondary siRNAs have been demonstrated in C. elegans, having different modifications at the 5′ends [49,50]. Primary siRNAs, which are the products of Dicer activity, have 5′-monophosphates and 3′-hydroxy groups whereas secondary RNAs are de novo synthesized by RDPs and have triphosphates at their 5′ end [49,50].…”

“…Full-length double-stranded RNA would be substrates for Dicer whereas the short RDP products proposedly would be directly incorporated into Argonaute complexes, guiding them towards complementary transcripts. Indeed, secondary siRNAs that are RDP products represent the most abundant class of small RNAs in C. elegans [49,50].…”

“…Primary siRNAs, which are the products of Dicer activity, have 5′-monophosphates and 3′-hydroxy groups whereas secondary RNAs are de novo synthesized by RDPs and have triphosphates at their 5′ end [49,50]. In plants, as well as the single-cell algae Chlamydomonas reinhardtii, the 3′-end of miRNA/siRNA is methylated and thereby protected from degradation [43,50,51]. This feature is not conserved in C. elegans or D. melanogaster where the 3′-ends investigated were unmodified and homologues of the responsible methyl-transferase do not seem to be conserved outside the kingdom of plants [52].…”