Although hundreds of evolutionarily conserved microRNAs have been discovered, the functions of most remain unknown. Here, we describe the embryonic spatiotemporal expression profile, transcriptional regulation, and loss-of-function phenotype of Drosophila miR-1 (DmiR-1). DmiR-1 RNA is highly expressed throughout the mesoderm of early embryos and subsequently in somatic, visceral, and pharyngeal muscles, and the dorsal vessel. The expression of DmiR-1 is controlled by the Twist and Mef2 transcription factors. DmiR-1 KO mutants, generated using ends-in gene targeting, die as small, immobilized second instar larvae with severely deformed musculature. This lethality is rescued when a DmiR-1 transgene is expressed specifically in the mesoderm and muscle. Strikingly, feeding triggers DmiR-1 KO -associated paralysis and death; starved first instar DmiR-1 KO larvae are essentially normal. Thus, DmiR-1 is not required for the formation or physiological function of the larval musculature, but is required for the dramatic post-mitotic growth of larval muscle.

Gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial as well as sporadic Parkinson's disease (PD) characterized by age-dependent dopaminergic neuron (DN) degeneration1 , 2. The molecular mechanism of LRRK2 action is not known. Here we show that LRRK2 interacts with the microRNA (miRNA) pathway to regulate protein synthesis. Drosophila e2f1 and dp mRNAs are translationally repressed by let-7 and miR-184*, respectively. Pathogenic LRRK2 antagonizes these miRNAs, leading to overproduction of E2F1/DP previously implicated in cell cycle and survival control 3 , and shown here to be critical for LRRK2 pathogenesis. Genetic deletion of let-7, antagomir-mediated blockage of let-7 and miR-184* action, transgenic expression of dp target protector, or replacing endogenous dp with a dp transgene non-responsive to let-7 all had similar toxic effects as pathogenic LRRK2. Conversely, increasing let-7 or miR-184* level attenuates pathogenic LRRK2 effects. LRRK2 associates with Drosophila Argonaute-1 (dAgo1) or human Argonaute-2 (hAgo2) of the RNA-induced silencing complex (RISC). In aged fly brain, dAgo1 protein level is negatively regulated by LRRK2. Further, pathogenic LRRK2 promotes the association of phospho-4E-BP1 with hAgo2. Our results implicate deregulated synthesis of E2F1/DP caused by miRNA pathway impairment as a key event in LRRK2 pathogenesis and suggest novel miRNA-based therapeutic strategies.Analyses of Dicer knockout mice have implicated the miRNA pathway in maintaining postmitotic neurons 4,5 . To test whether LRRK2 might affect miRNA function, we generated an in vivo EGFP reporter with let-7-binding sites in the 3′UTR (EGFP-let-7-3′UTR). Loss of one copy of Drosophila dicer-1 or argonaute 1 (ago1) genes of the miRNA pathway enhanced reporter expression (Fig.1a), suggesting that this reporter faithfully monitors endogenous miRNA activity. Da-Gal4-directed ubiquitous or TH-Gal4-directed DN-specific co-expression of pathogenic dLRRK(I1915T) or hLRRK2(G2019S) all stimulated reporter expression, more so than WT proteins (Fig.1a, 1b). This effect correlated with differential toxicity 6 , rather than expression levels of the proteins, since the pathogenic proteins were actually expressed at lower levels (Fig.2a). dLRRK RNAi led to decreased reporter expression, indicating that endogenous dLRRK also impacts miRNA function (Fig.1a). The kinase-dead dLRRK(3KD) did not affect reporter expression (Fig.1a , s1a). This result, together with the observation that I1915T and G2019S mutations augmented kinase activity 7,8 , suggests that the effect of LRRK2 is kinase-dependent. gfp mRNA levels were not affected (Fig.s1b,c). Moreover, the expression of an EGFP transgene without let-7-

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The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). We find that the let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. We find that the function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-toadult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which let-7-C promotes adult behavior.Supplemental material is available at http://www.genesdev.org.Received March 10, 2008; revised version accepted April 11, 2008. Mutations in heterochronic genes in Caenorhabditis elegans cause cells in particular lineages to express their stage-specific fates earlier or later than normal (Ambros and Horvitz 1984). Detailed analysis of these genes has revealed a regulatory pathway of heterochronic genes that specifies the timing of cellular development in diverse cell types and thereby ensures a coordinated schedule of developmental events throughout the worm (for review, see Rougvie 2005; Moss 2007). The existence of the heterochronic gene pathway in worms and the conservation of some of its components through animal evolution suggest that functionally analogous pathways could also coordinate developmental timing in higher organisms . Two of these highly conserved components of the heterochronic pathway, let-7 and lin-4, are microRNAs (miRNAs), a class of small RNAs that post-transcriptionally modulate the expression of target transcripts (for review, see Jackson and Standart 2007). The sequences and developmentally regulated expression profiles of let-7 and lin-4 are conserved among diverse bilaterians Sempere et al. 2003). For example, Drosophila let-7 and miR-125 (fly lin-4) are robustly up-regulated during metamorphosis, as is another highly conserved miRNA, miR-100 Sempere et al. 2002Sempere et al. , 2003Bashirullah et al. 2003). All three of these ancient miRNAs are encoded in a 1-kb region of the Drosophila genome ( Fig. 1; Sempere et al. 2003), and their clustered organization has been conserved and duplicated in vertebrates (Supplemental Fig. S1; Sempere et al. 2003;Prochnik et al. 2007). These findings suggest that miR-100, let-7, and miR-125 coordinately control gene expression to regulate developmental timing in animals.To test this hypothesis, we analyzed the roles of miR-100, let-7, and miR-125 in Drosophila and find that these miRNAs are required for normal adult behavior, suggesting roles in neural development and/or function. let-7 in particular is required for remodeling of the fly neuromusculature during the larval-to-adult transition, confirming that a general developmental timing function of let-7 has been evolutionarily conserved from worms to flies. Results and DiscussionThe clus...

Localization of Filamin to nascent ring canals is necessary for the recruitment of actin filaments. We propose that Filamin links filamentous actin to the plasma membrane of the ring canal. Although loss of Filamin in human cells supports a role for Filamin in organizing orthogonal actin arrays at the cell cortex, the cheerio mutant provides the first evidence that Filamin is required in membrane-associated parallel actin bundles, such as those found in ring canals, contractile rings and stress fibers.