The FT gene integrates several external and endogenous cues controlling flowering, including information on day length. A complex of the mobile FT protein and the bZIP transcription factor FD in turn has a central role in activating genes that execute the switch from vegetative to reproductive development. Here we reveal that microRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes not only act downstream of FT/FD, but also define a separate endogenous flowering pathway. High levels of miR156 in young plants prevent precocious flowering. A subsequent day length-independent decline in miR156 abundance provides a permissive environment for flowering and is paralleled by a rise in SPL levels. At the shoot apex, FT/FD and SPLs converge on an overlapping set of targets, with SPLs directly activating flower-promoting MADS box genes, providing a molecular substrate for both the redundant activities and the feed-forward action of the miR156/SPL and FT/FD modules in flowering control.

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes [1][2][3][4][5][6][7] . These join Argonaute-1 to regulate gene expression 8,9 . A second endogenous small RNA class, the Piwiinteracting RNAs, bind Piwi proteins and suppress transposons 10,11 . Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of singlestranded RNAs 12,13 . Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious 1,4,5 rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.Drosophila melanogaster expresses five Argonaute proteins, which segregate into two classes. The Piwi proteins (Piwi, Aubergine and AGO3) are expressed in gonadal tissues and act with Piwi-interacting RNAs (piRNAs) to suppress mobile genetic elements 10,11 . The Argonaute class contains AGO1 and AGO2. AGO1 binds microRNAs (miRNAs) and regulates gene expression 8,9 . The endogenous binding partners of AGO2 have remained enigmatic.We generated transgenic flies expressing epitope-tagged AGO2 under the control of its endogenous promoter. Tagged AGO2 localized to the cytoplasm of germline and somatic cells of the ovary (Supplementary Fig. 1). Immunoprecipitated AGO2-associated RNAs differed in their mobility from those bound to AGO1 (Fig. 1a). Deep sequencing of small RNAs from AGO1 and AGO2 complexes yielded 2,094,408 AGO1-associated RNAs and 916,834 AGO2-associated RNAs from Schneider (S2) cells, and 455,227 AGO2-associated RNAs from ovaries that matched perfectly to the Drosophila genome. We also sequenced three libraries derived from 18-29-nucleotide RNAs (936,833 sequences from wild-type ovaries, 1,042,617 sequences from Dicer-2 (Dcr-2) mutant ovaries, and 1,946,339 sequences from loquacious (loqs) mutant ovaries) and an 18-24-nucleotide library from wild-type testes (522,848 sequences). Finally, we added to our analysis 92,363 published sequences derived from 19-26-nucleotide RNAs from S2 cells 15 . We noted that among the ,50% of AGO2-associated RNAs from S2 cells that did not match the genome, ,17% matched the flock house virus (FHV), a pathogenic RNA virus and reported target for RNAi in flies 16,17 . These probably arose because o...

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.