Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3′ exons generate 21-nt, Dicer-2 (Dcr-2)–dependent, 3′-end modified siRNAs. The processing of cis-NATs by RNA interference (RNAi) seems to be actively restricted, and the selected loci are enriched for nucleic acid–based functions and include Argonaute-2 (AGO2) itself. Second, we report that extended intervals of the thickveins and klarsicht genes generate exceptionally abundant siRNAs from both strands. These siRNA clusters derive from atypical cis-NAT arrangements involving introns and 5′ or internal exons, but their biogenesis is similarly Dcr-2– and AGO2-dependent. These newly recognized siRNA pathways broaden the scope of regulatory networks mediated by small RNAs.
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