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In Drosophila melanogaster, the small interfering RNA (siRNA) pathway is triggered by exogenous double-stranded RNA (dsRNA) or upon viral infection. This pathway requires Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) called R2D2. A potentially distinct siRNA pathway, which requires Dcr-2 in association with a different dsRBP, called Loquacious (Loqs), is activated by endogenous dsRNA derived from transposons, structured loci and overlapping transcripts. Here we show that different sources of dsRNA enter a common siRNA pathway that requires R2D2 and Loqs. R2D2 and loqs mutants show impaired silencing triggered by injection of exogenous dsRNA or by artificial and natural expression of endogenous dsRNA. In addition, we show that these dsRBPs function sequentially and nonredundantly in collaboration with Dcr-2. Loqs is primarily required for dsRNA processing, whereas R2D2 is essential for the subsequent loading of siRNAs into effector Ago–RISC complexes. [ABSTRACT FROM AUTHOR]

Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm. [ABSTRACT FROM AUTHOR]

Summary: The microRNA miR-7 is perfectly conserved from annelids to humans, and yet some of the genes that it regulates in Drosophila are not regulated in mammals. We have explored the role of lineage restricted targets, using Drosophila, in order to better understand the evolutionary significance of microRNA-target relationships. From studies of two well characterized developmental regulatory networks, we find that miR-7 functions in several interlocking feedback and feedforward loops, and propose that its role in these networks is to buffer them against perturbation. To directly demonstrate this function for miR-7, we subjected the networks to temperature fluctuation and found that miR-7 is essential for the maintenance of regulatory stability under conditions of environmental flux. We suggest that some conserved microRNAs like miR-7 may enter into novel genetic relationships to buffer developmental programs against variation and impart robustness to diverse regulatory networks. [Copyright &y& Elsevier]

Abstract: The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure–function relationship of Dicer, which plays a critical role in the siRNA pathway. [Copyright &y& Elsevier]

MicroRNAs (miRNAs) are small, endogenously expressed RNAs that regulate mRNAs post-transcriptionally. The class of miRNA genes, like other gene classes, should experience birth, death and persistence of its members. We carried out deep sequencing of miRNAs from three species of Drosophila, and obtained 107,000 sequences that map to no fewer than 300 loci that were not previously known. We observe a large class of miRNA genes that are evolutionarily young, with a rate of birth of 12 new genes per million years (Myr). Most of these new miRNAs originated from non-miRNA sequences. Among the new genes, we estimate that 96% disappeared quickly in the course of evolution; only 4% of new miRNA genes were retained by natural selection. Furthermore, only 60% of these retained genes became integrated into the transcriptome in the long run (60 Myr). This small fraction (2.5%) of surviving miRNAs may later on become moderately or highly expressed. Our results suggest that there is a high birth rate of new miRNA genes, accompanied by a comparably high death rate. The estimated net gain of long-lived miRNA genes, which is not strongly affected by either the depth or the breadth (number of tissues) of sequencing, is 0.3 genes per Myr in Drosophila. [ABSTRACT FROM AUTHOR]

Because of the resemblance of many epithelial tissues to densely packed soap bubbles, it has been suggested that surface minimization, which drives soap bubble packing, could be governing cell packing as well. We test this by modeling the shape of the cells in a Drosophila retina ommatidium. We use the observed configurations and shapes in wild-type flies, as well as in flies with different numbers of cells per ommatidia, and mutants with cells where E- or N-cadherin is either deleted or misexpressed. We find that surface minimization is insufficient to model the experimentally observed shapes and packing of the cells based on their cadherin expression. We then consider a model in which adhesion leads to a surface increase, balanced by cell cortex contraction. Using the experimentally observed distributions of E- and N-cadherin, we simulate the packing and cell shapes in the wild-type eye. Furthermore, by changing only the corresponding parameters, this model can describe the mutants with different numbers of cells or changes in cadherin expression. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3' untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, we compared the proteome from wild-type oocytes with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3' untranslated region for a subset of miRNAs, based on computer modeling The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization. [ABSTRACT FROM AUTHOR]

The RNase III enzyme Dicer processes RNA into si-RNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, Whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-I but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRlSCs and miRISCs are different with respect to Dicers in Drosophila. [ABSTRACT FROM AUTHOR]

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi. [ABSTRACT FROM AUTHOR]

Inappropriate activation of downstream target genes by the oncoprotein ?-catenin is implicated in development of numerous human cancers. ?-catenin and its fruitfly counterpart Armadillo act as a coactivator in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. Here we report a conserved nuclear protein, named Chibby, which was identified in a screen for proteins that directly interact with the C-terminal region of ?-catenin. In mammalian cultured cells we demonstrate that Chibby inhibits ?-catenin-mediated transcriptional activation by competing with Lef-1 to bind to ?-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo. [ABSTRACT FROM AUTHOR]

Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. We find that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. We demonstrate the universality of this phenomenon by eliminating the entire microRNA family of repressors and find that development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, we find that lowering metabolism suppresses the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. We experimentally show that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle. • Cellular metabolic rate controls the tempo of development • Multiple weak repressors allow GRN dynamics to adjust to a variable tempo • Slow metabolism renders individual repressors redundant • microRNAs become dispensable for development in the context of slower metabolism microRNAs become dispensable for development in the context of slower metabolism. [ABSTRACT FROM AUTHOR]

A correction to the article "Silencing by Small RNAs Is Linked to Endosomal Trafficking," that was published online in the August 16, 2009 issue is presented.