microRNAs (miRNAs) function in posttranscriptional control of gene expression through RISC (RNA-induced silencing complex)–mediated inhibition of translation, often accompanied by destabilization of the targeted mRNAs

control of gene expression through RISC (RNA-induced silencing complex)–mediated inhibition of translation, often accompanied by destabilization of the targeted mRNAs (for review, see Filipowicz et al. 2005; Valencia-Sanchez et al. 2006). miRNA biogenesis involves several coordinated processing and intracellular transport steps, some of which appear to be controlled during development and differentiation or in response to environmental stimuli (for review, see Bartel 2004; Du and Zamore 2005). These steps include (1) synthesis of primary transcripts containing one or more miRNAs, (2) processing of these transcripts by the RNase-III-like endonuclease Drosha (and associated proteins) to produce approximately 65to 75-nucleotide-long incompletely base-paired hairpin RNAs (pre-miRNAs), (3) export of pre-miRNAs from the nucleus to the cytoplasm by the export factor exportin-5 (Exp5), (4) cleavage of the pre-miRNAs in the cytoplasm by a second doublestrand-specific RNase-III-like processing enzyme, Dicer, into duplexes about 22 nucleotides long, and (5) selection of one strand of the product duplex as the miRNA and incorporation into a ribonucleoprotein complex, the RISC (for review, see Murchison and Hannon 2004; Kim 2005). Here we describe our studies on the activities, substrate specificities, and developmental control of two of the key proteins that participate in this process, Exp5 and Dicer (steps 3 and 4). We have asked what features of pre-miRNA substrates contribute to their export by Exp5 and their processing by Dicer. These hairpin RNAs have imperfectly base-paired stem regions about 22 nucleotides long, flanked by free 5′ and 3′ ends and an unpaired loop region. To test the importance of these structural elements in export and processing, we prepared P-labeled RNA substrates (by in vitro transcription; Grimm et al. 1997) in which these elements were either altered or deleted. We then monitored the fates of the variant RNAs in vivo, using microinjected Xenopus oocytes and embryos, or in vitro, using human recombinant Dicer.