Transgenic inhibitors of RNA interference in Drosophila

RNA silencing functions as an adaptive antiviral defense in both plants and animals. In turn, viruses commonly encode suppressors of RNA silencing, which enable them to mount productive infection. These inhibitor proteins may be exploited as reagents with which to probe mechanisms and functions of RNA silencing pathways. In this report, we describe transgenic Drosophila strains that allow inducible expression of the viral RNA silencing inhibitors Flock House virus-B2, Nodamura virus-B2, vaccinia virus-E3L, influenza A virus-NS1 and tombusvirus P19. Some of these, especially the B2 proteins, are effective transgenic inhibitors of double strand RNA-induced gene silencing in flies. On the other hand, none of them is effective against the Drosophila microRNA pathway. Their functional selectivity makes these viral silencing proteins useful reagents with which to study biological functions of the Drosophila RNA interference pathway.     

Analysis of Drosophila 26 S proteasome using RNA interference.

We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.     

Drosophila development: RNA interference ab ovo

A novel protein required for RNA interference in Drosophila, Armitage, was identified in a screen for genes involved in embryonic axis formation. In armitage mutants, oocyte polarity and the regulation of oskar mRNA translation are impaired, suggesting that RNA silencing regulates the first steps of Drosophila development.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

RNA interference has a role in regulating Drosophila telomeres

Unlike many other organisms, Drosophila maintains its telomeres by the transposition of retrotransposons to chromosome ends. Recent work shows that proteins in the RNA interference pathway specifically regulate the expression of these retrotransposons and frequency of transposition in germline cells, but do not affect retrotransposon expression or telomere function in the soma.     

Recurrent adaptation in RNA interference genes across the Drosophila phylogeny

RNA interference (RNAi) is quickly emerging as a vital component of genome organization, gene regulation, and immunity in Drosophila and other species. Previous studies have suggested that, as a whole, genes involved in RNAi are under intense positive selection in Drosophila melanogaster. Here, we characterize the extent and patterns of adaptive evolution in 23 known Drosophila RNAi genes, both within D. melanogaster and across the Drosophila phylogeny. We find strong evidence for recurrent protein-coding adaptation at a large number of RNAi genes, particularly those involved in antiviral immunity and defense against transposable elements. We identify specific functional domains involved in direct protein-RNA interactions as particular hotspots of recurrent adaptation in multiple RNAi genes, suggesting that targeted coadaptive arms races may be a general feature of RNAi evolution. Our observations suggest a predictive model of how selective pressures generated by evolutionary arms race scenarios may affect multiple genes across protein interaction networks and other biochemical pathways.     

Parasite-specific immune response in adult Drosophila melanogaster: a genomic study

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite-specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence.     

Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster

The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.     

Widespread disruption of genomic imprinting in adult interspecies mouse (Mus) hybrids

Mammalian interspecies hybrids exhibit parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit opposite phenotypes, especially in growth. This was also observed in hybridization with the genus Mus. These parent-of-origin effects suggested that imbalance in the expression of imprinted genes, which are expressed differentially, depending on their transmission through the maternal or paternal germline, and/or differential loss-of-imprinting (LOI) could underlie these opposite growth phenotypes in reciprocal mammalian hybrids. Here we report that tissue-specific LOI occurs in adult Mus hybrids. Contrary to expectations, LOI patterns were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Bisulfite sequencing revealed that reactivation of maternal alleles of Peg3 and Snrpn in specific tissues was accompanied by partial demethylation at their potential imprinting control regions. We propose that abnormal reprogramming after fertilization and during preimplantation development is in part responsible for hybrid dysgenesis, for which a strong epigenetic basis has been demonstrated.     

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila

BACKGROUND: The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. RESULTS: When expressed under the control of a heat-inducible promoter, dsRNA interfered efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generated defects in larval molting and metamorphosis, resulting in animals that failed to pupariate or prepupae that died with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor had no effect on puparium formation, but led to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that expressed either EcR or betaFTZ-F1 dsRNA showed defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. CONCLUSIONS: These results confirm and extend our understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila.     

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis.     

Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.