In plants, cis-acting siRNAs (casiRNAs) originate from transposons, repetitive elements, and tandem repeats such as 5S rRNA genes, and comprise the bulk of endo-siRNAs44 (FIG. 2). CasiRNAs are predominantly 24 nt long and methylated by HEN1. Their accumulation requires DCL3 and the RNA polymerases, RDR2 and POL IV, and either AGO6 (primarily) or AGO4, which act redundantly44,51–60. CasiRNAs promote heterochromatin formation by directing DNA methylation and histone modification of the loci from which they originate38,44,51,52,61–63.

The current model for piRNA biogenesis was inferred from the sequences of piRNAs bound to Piwi, Aubergine and Ago3176,183. piRNAs bound to Piwi and Aubergine are typically antisense to transposon mRNAs, whereas Ago3 is loaded with piRNAs corresponding to the transposon mRNAs themselves (FIG. 1). Moreover, the first 10 nucleotides of antisense piRNAs are frequently complementary to the sense piRNAs found in Ago3. This unexpected sequence complementarity has been proposed to reflect a feed-forward amplification mechanism—”piRNA ping-pong”—that is activated only upon transposon mRNA transcription (FIG. 4)176,183. A similar amplification loop has been inferred from high throughput piRNA sequencing in vertebrates, implying its conservation through evolution162,171. Many aspects of the ping-pong model remain speculative. Why Ago3 appears to bind only sense piRNAs derived from transposon mRNAs is unknown. An untested idea is that different forms of RNA Pol II transcribe primary piRNA transcripts and transposon mRNAs and that the specialized RNA Pol II that transcribes the primary piRNA precursor recruits Piwi and Aub, but not Ago3. How the 3′ ends of piRNAs are made is also not known.

Early examples of RNAi were triggered by exogenous dsRNA. In these cases, long, exogenous dsRNA is cleaved into double-stranded siRNAs by Dicer (Dcr), a dsRNA-specific RNase III family ribonuclease7 (FIG. 1). siRNA duplexes produced by Dicer comprise two ~21 nt strands, each bearing a 5′ phosphate and 3′ hydroxyl group, paired so as to leave 3′, two-nucleotide overhangs5,8,9. The strand that directs silencing is called the guide, whereas the other strand, which is ultimately destroyed, is the passenger. Target regulation by siRNAs is mediated by the RNA-induced silencing complex (RISC), the generic name for an Argonaute-small RNA complex6. In addition to an Argonaute protein and a small RNA guide, RISC may also contain auxiliary proteins that extend or modify its function, for example, proteins that re-direct the target mRNA to a site of general mRNA degradation10.

Fly endo-siRNAs derive from transposons, heterochromatic sequences, intergenic regions, long RNA transcripts with extensive structure, and, most interestingly, from mRNAs (FIG. 3). Expression of transposon mRNAs increases in both dcr-2 and ago2 mutants, implicating an endogenous RNAi pathway in the silencing of transposons in flies, as reported previously for C. elegans82,83. siRNAs derived from mRNAs are >10 times more likely to come from regions predicted to produce overlapping, convergent transcripts than expected by chance76, suggesting that endo-siRNAs originate from endogenous dsRNA formed when these complementary transcripts pair.