MicroRNAs regulate mRNA stability and translation in plants, green algae and animals [1
]. Originally discovered in Caenorhabditis elegans
, these ~22 nucleotide (nt) small RNAs regulate development and physiology, and have been implicated in diseases such as cancer, diabetes, and viral infection [3
]. Loss of proteins required for the production or function of miRNAs typically results in severe developmental defects or lethality.
miRNA genes are generally transcribed by RNA polymerase II to generate 52 capped and 32 polyadenylated primary miRNAs (pri-miRNAs) that are then sequentially processed into mature miRNA duplexes [6
]. Pri-miRNAs contain one or more characteristic stem-loops that are recognized and cleaved by the nuclear RNase III enzyme Drosha [7
] to generate ~70 nt long precursor miRNAs (pre-miRNAs) [8
]. Pre-miRNAs comprise a single-stranded loop and a partially base-paired stem whose termini bear the hallmarks of RNase III processing: a two-nucleotide 3′ overhang, a 52 phosphate and a 32 hydroxyl group.
Nuclear pre-miRNAs are exported by Exportin 5 to the cytoplasm, where the RNase III enzyme Dicer liberates ~22 nt mature miRNA/ miRNA* duplexes from the pre-miRNA stem [9
]. Like all Dicer products, miRNA duplexes contain two-nucleotide 32 overhangs, 52 phosphate and 32 hydroxyl groups. In flies, Dicer-1 cleaves pre-miRNAs to miRNAs, while Dicer-2 converts long dsRNA into small interfering RNAs (siRNAs), which direct RNA interference (RNAi), a distinct small RNA silencing pathway required for host defense against viral infection and somatic transposon mobilization, as well as gene silencing triggered by exogenous dsRNA [13
miRNA duplexes assemble into Argonaute proteins to form the precursor RNA-Induced Silencing Complex (pre-RISC), a process uncoupled from small RNA production [15
]. In flies, miRNAs typically bind to Argonaute1 and siRNAs to Argonaute2 [16
]. During RISC assembly one of the two strands of a miRNA duplex is selectively retained to form an active silencing complex. Strand selection is determined by the relative thermodynamic stability of the duplex ends, the identity of the 52 nucleotides, as well as the structure and length of the miRNA duplex [15
]. In mature RISC, a single-stranded miRNA directs Ago1 to bind partially complementary sequences, typically within the 32 untranslated region (32 UTR) of mRNAs [1
]. RISC-binding represses mRNA expression by accelerating its decay or inhibiting its translation [17
Here, we report that more than one quarter of all miRNAs in Drosophila S2 cells are trimmed after their loading into Ago1, a process that can be recapitulated in cell extracts and that we can detect in vivo in flies. Trim ming of miRNAs is mediated by the Mg2+-dependent 32-to-52 exoribonuclease Knabber (CG9247), a member of the DEDD family of exonucleases. Knabber activity is required to trim Ago1-loaded miRNAs, and miRNA trimming enhances target RNA repression. Knabber is required for normal fly development. Our results show that the 32 ends of miRNAs are not simply defined by the RNase III enzymes Drosha and Dicer-1, but undergo exonucleolytic reshaping after their loading into Ago1. Thus, the previously described heterogeneity of miRNA 32 ends reflects mainly active trimming, rather than sloppy precursor processing.