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Originally published in Science Express on 7 December 2006
Science 12 January 2007:
Vol. 315. no. 5809, pp. 244 - 247
DOI: 10.1126/science.1136699
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Reports

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

Titia Sijen,* Florian A. Steiner,* Karen L. Thijssen, Ronald H. A. Plasterk{dagger}

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.

Hubrecht Laboratory (NIOB-KNAW), Uppsalalaan 8, 3584 CT, the Netherlands.

* These authors contributed equally to this work.

{dagger} To whom correspondence should be addressed. E-mail: plasterk{at}niob.knaw.nl

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