MicroRNAs (miRNAs) are initially expressed as long transcripts that are processed in the nucleus to yield ∼65-nucleotide (nt) RNA hairpin intermediates, termed premiRNAs, that are exported to the cytoplasm for additional processing to yield mature, ∼22-nt miRNAs. Here, we demonstrate that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor. Short hairpin RNAs, artificial pre-miRNA analogs used to express small interfering RNAs, also depend on Exportin-5 for nuclear export. Together, these findings define an additional cellular cofactor required for miRNA biogenesis and function. Received October 7, 2003; revised version accepted October 28, 2003. MicroRNAs (miRNAs) are ∼22-nucleotide (nt) noncoding RNAs observed in a wide range of eukaryotes. Over 200 genomically encoded miRNAs have been identified in several different species (Ambros 2003). Although few animal miRNAs have been assigned a function to date, the Caenorhabditis elegans let-7 and lin-4 miRNAs and the miR-14 and bantam miRNAs found in Drosophila all repress the expression of mRNAs bearing partially complementary target sites (Ambros 2003). In C. elegans, let-7 and lin-4 expression is developmentally regulated, and loss of let-7 or lin-4 function results in disruption of normal larval development due to the inappropriate expression of their mRNA targets (Lee et al. 1993;Reinhart et al. 2000). Consistent with a similar role for miRNAs in vertebrate development, many murine miRNAs are also expressed in a developmentally regulated or tissue-specific manner (Lagos-Quintana et al. 2002;Houbaviy et al. 2003).The miRNAs are initially expressed as part of an imperfect RNA hairpin of ∼80 nt in length that in turn forms part of a longer initial transcript termed a primary miRNA (pri-miRNA; Lee et al. 2002). The first step in miRNA biogenesis is the nuclear excision of the upper part of this RNA hairpin to give the ∼65-nt pre-miRNA intermediate (Lee et al. 2002;. This processing step is performed by human RNAse III, also called 'Drosha' (Lee et al. 2003). The pre-miRNA intermediate, which in the case of human miR-30 consists of a 63-nt hairpin bearing a 2-nt 3Ј overhang, is then exported to the cytoplasm by a currently unknown mechanism. Once there, the pre-miRNA is processed by a second RNAse III family member called 'Dicer' to give the mature ∼22-nt miRNA (Grishok et al. 2001;Hutvágner et al. 2001;Ketting et al. 2001). The miRNA is then incorporated into the RNA-induced silencing complex (RISC), where it functions to guide RISC to appropriate mRNA targets (Hammond et al. 2000;Martinez et al. 2002;Mourelatos et al. 2002;Schwarz et al. 2002).In (Brummelkamp et al. 2002;Paddison et al. 2002). The shRNAs that have been described generally feature perfect 19-29-base pair (bp) stems with small terminal loops and a 2-nt 3Ј overhang, consisting of two "U" residues, that results from termination of pol III transcription. Therefore, th...

Recent studies show that AMPA receptor (-R) trafficking is important in synaptic plasticity. However, the signaling controlling this trafficking is poorly understood. Small GTPases have diverse neuronal functions and their perturbation is responsible for several mental disorders. Here, we examine the small GTPases Ras and Rap in the postsynaptic signaling underlying synaptic plasticity. We show that Ras relays the NMDA-R and CaMKII signaling that drives synaptic delivery of AMPA-Rs during long-term potentiation. In contrast, Rap mediates NMDA-R-dependent removal of synaptic AMPA-Rs that occurs during long-term depression. Ras and Rap exert their effects on AMPA-Rs that contain different subunit composition. Thus, Ras and Rap, whose activity can be controlled by postsynaptic enzymes, serve as independent regulators for potentiating and depressing central synapses.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R trafficking is unknown. Here we show that low levels of Ras activity stimulate extracellular signal-regulated kinase kinase (MEK)-p42/44 MAPK (extracellular signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity stimulate additional Pi3 kinase (Pi3K)-protein kinase B (PKB) signaling, each accounting for ∼50% of the potentiation during long-term potentiation (LTP). Spontaneous neural activity stimulates the Ras-MEK-ERK pathway that drives GluR2L into synapses. In the presence of neuromodulator agonists, neural activity also stimulates the Ras-Pi3K-PKB pathway that drives GluR1 into synapses. Neuromodulator release increases with increases of vigilance. Correspondingly, Ras-MEK-ERK activity in sleeping animals is sufficient to deliver GluR2L into synapses, while additional increased Ras-Pi3K-PKB activity in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras signaling, which specifies downstream MEK-ERK and Pi3K-PKB pathways, differentially control GluR2L-and GluR1-dependent synaptic plasticity.[Keywords: Ras; Erk; Pi3 kinase; Src; synaptic plasticity; neuromodulators; behavioral states] Supplemental material is available at http://www.genesdev.org.