Titia Sijen ¹, Florian A. Steiner ¹, Karen L. Thijssen¹ , and Ronald H. A. Plasterk ^1, *

¹ Hubrecht Laboratory (NIOB-KNAW), Uppsalalaan 8, 3584 CT, the Netherlands.

* To whom correspondence should be addressed.
Ronald H. A. Plasterk , E-mail: plasterk@niob.knaw.nl

In C. elegans, an effective RNA interference (RNAi) response requires the production of secondary 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 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.

http://www.sciencemag.org/cgi/content/full/sci;1136699/DC1

Materials and Methods
Figs. S1 to S4
Tables S1 and S2
References

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".