Abstract
microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs.
Highlights
MicroRNAs act as RNA guides by binding to complementary sites on target mRNAs to regulate gene expression at the post-transcriptional level in plants and animals [1À12], much as small interfering RNAs do in the RNA interference (RNAi) pathway [13À15]
We have used RNAi-based reverse-genetic methods [61] to screen a list of Drosophila double-stranded RNA-binding domain (dsRBD) proteins [62] for a protein(s) that has an effect on miRNA biogenesis in Drosophila S2 cells and found a novel protein equipped with three dsRBDs, originally dubbed CG6866, which has a role in premiRNA processing
Another Argonaute protein AGO2-associated complex showed no such activity, which is consistent with our previous finding that the AGO2-associated complex does not contain Dicer-1 [60]. Considered together, these results showed that Dicer-1 and Loqs form a functional complex that mediates the genesis of mature miRNAs from pre-miRNAs, and suggested that the resultant mature miRNAs are loaded onto an AGO1-associated complex, which probably is miRNAassociated RNA-induced silencing complex (RISC) [60], through specific interaction of AGO1 with Dicer-1 and Loqs
Summary
MicroRNAs (miRNAs) act as RNA guides by binding to complementary sites on target mRNAs to regulate gene expression at the post-transcriptional level in plants and animals [1À12], much as small interfering RNAs (siRNAs) do in the RNA interference (RNAi) pathway [13À15]. Recent studies have revealed that miRNAs regulate a large fraction of the protein-coding genes [32,33,34]. MiRNA maturation begins with cleavage of the pri-miRNAs by the nuclear RNase III Drosha [36,37,38] to release approximately 70-nucleotide hairpin-shaped structures, called precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported to the cytoplasm by the protein Exportin 5, which recognizes the two-nucleotide 39 overhang that is a signature of RNase IIImediated cleavage [39,40,41]. Pre-miRNAs are subsequently cleaved by a second RNase III enzyme, Dicer, into approximately 22-nucleotide miRNA duplexes, with an end structure characteristic of RNase III cleavage [42,43,44]. One of the two strands is predominantly transferred to the RNA-induced silencing complex (RISC) [45], which mediates either cleavage of the target mRNA or translation silencing, depending on the complementarity of the target [46] by a mechanism that remains unclear [47]
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