The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs.

G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition.

BACKGROUND: Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. RESULTS: Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. CONCLUSIONS: Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Transcriptional profile of muscle following acute induction of symptoms in a mouse model of Kennedy's disease/spinobulbar muscular atrophy.

BACKGROUND: Kennedy's disease/Spinobulbar muscular atrophy (KD/SBMA) is a degenerative neuromuscular disease affecting males. This disease is caused by polyglutamine expansion mutations of the androgen receptor (AR) gene. Although KD/SBMA has been traditionally considered a motor neuron disease, emerging evidence points to a central etiological role of muscle. We previously reported a microarray study of genes differentially expressed in muscle of three genetically unique mouse models of KD/SBMA but were unable to detect those which are androgen-dependent or are associated with onset of symptoms. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we examined the time course and androgen-dependence of transcriptional changes in the HSA-AR transgenic (Tg) mouse model, in which females have a severe phenotype after acute testosterone treatment. Using microarray analysis we identified differentially expressed genes at the onset and peak of muscle weakness in testosterone-treated Tg females. We found both transient and persistent groups of differentially expressed genes and analysis of gene function indicated functional groups such as mitochondrion, ion and nucleotide binding, muscle development, and sarcomere maintenance. CONCLUSIONS/SIGNIFICANCE: By comparing the current results with those from the three previously reported models we were able to identify KD/SBMA candidate genes that are androgen dependent, and occur early in the disease process, properties which are promising for targeted therapeutics.

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

BACKGROUND: Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. RESULTS: To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. CONCLUSIONS: Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Prediction of Drosophila melanogaster gene function using Support Vector Machines.

BACKGROUND: While the genomes of hundreds of organisms have been sequenced and good approaches exist for finding protein encoding genes, an important remaining challenge is predicting the functions of the large fraction of genes for which there is no annotation. Large gene expression datasets from microarray experiments already exist and many of these can be used to help assign potential functions to these genes. We have applied Support Vector Machines (SVM), a sigmoid fitting function and a stratified cross-validation approach to analyze a large microarray experiment dataset from Drosophila melanogaster in order to predict possible functions for previously un-annotated genes. A total of approximately 5043 different genes, or about one-third of the predicted genes in the D. melanogaster genome, are represented in the dataset and 1854 (or 37%) of these genes are un-annotated. RESULTS: 39 Gene Ontology Biological Process (GO-BP) categories were found with precision value equal or larger than 0.75, when recall was fixed at the 0.4 level. For two of those categories, we have provided additional support for assigning given genes to the category by showing that the majority of transcripts for the genes belonging in a given category have a similar localization pattern during embryogenesis. Additionally, by assessing the predictions using a confidence score, we have been able to provide a putative GO-BP term for 1422 previously un-annotated genes or about 77% of the un-annotated genes represented on the microarray and about 19% of all of the un-annotated genes in the D. melanogaster genome. CONCLUSIONS: Our study successfully employs a number of SVM classifiers, accompanied by detailed calibration and validation techniques, to generate a number of predictions for new annotations for D. melanogaster genes. The applied probabilistic analysis to SVM output improves the interpretability of the prediction results and the objectivity of the validation procedure.

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics.

BACKGROUND: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. RESULTS: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. CONCLUSIONS: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS's mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Synthetic antibodies as tools to probe RNA-binding protein function.

RNA-binding proteins (RBPs) have essential roles in post-transcriptional regulation of gene expression. They bind sequence elements in specific mRNAs and control their splicing, transport, localization, translation, and stability. A complete understanding of RBP function requires identification of the target RNAs that an RBP regulates, the mechanisms by which the RBP regulates these targets, and the biological consequences for the cell in which these transactions occur. Antibodies are key tools in such studies: first, mRNA targets of RBPs can be identified by co-immunoprecipitation of RBPs with their associated RNAs followed by microarray analysis or sequencing; second, partner proteins can be identified by immunoprecipitation of the RBP followed by mass spectrometry; third, the mechanisms and functions of RBPs can be inferred from loss-of-function studies employing antibodies that block RBP-RNA interactions. One potentially powerful approach to making antibodies for such studies is the generation of synthetic antibodies using phage display, which involves in vitro selection using a human-designed antibody library to generate antibodies that recognize a target protein. Using two well-characterized Drosophila RNA-binding proteins, Staufen and Smaug, for proof-of-principle, we demonstrate that synthetic antibodies can be generated and used either to perform RNA-coimmunoprecipitations (RIPs) to identify RBP-bound mRNAs, or to block RBP-RNA interactions. Given that synthetic antibody selection protocols are amenable to high-throughput antibody production, these results demonstrate that synthetic antibodies can be powerful tools for genome-wide studies of RBP function.

Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells.

BACKGROUND: During the maternal-to-zygotic transition (MZT) vast changes in the embryonic transcriptome are produced by a combination of two processes: elimination of maternally provided mRNAs and synthesis of new transcripts from the zygotic genome. Previous genome-wide analyses of the MZT have been restricted to whole embryos. Here we report the first such analysis for primordial germ cells (PGCs), the progenitors of the germ-line stem cells. RESULTS: We purified PGCs from Drosophila embryos, defined their proteome and transcriptome, and assessed the content, scale and dynamics of their MZT. Transcripts encoding proteins that implement particular types of biological functions group into nine distinct expression profiles, reflecting coordinate control at the transcriptional and posttranscriptional levels. mRNAs encoding germ-plasm components and cell-cell signaling molecules are rapidly degraded while new transcription produces mRNAs encoding the core transcriptional and protein synthetic machineries. The RNA-binding protein Smaug is essential for the PGC MZT, clearing transcripts encoding proteins that regulate stem cell behavior, transcriptional and posttranscriptional processes. Computational analyses suggest that Smaug and AU-rich element binding proteins function independently to control transcript elimination. CONCLUSIONS: The scale of the MZT is similar in the soma and PGCs. However, the timing and content of their MZTs differ, reflecting the distinct developmental imperatives of these cell types. The PGC MZT is delayed relative to that in the soma, likely because relief of PGC-specific transcriptional silencing is required for zygotic genome activation as well as for efficient maternal transcript clearance.

Divergent transcriptomic responses to repeated and single cold exposures in Drosophila melanogaster.

Insects in the field are exposed to multiple bouts of cold, and there is increasing evidence that the fitness consequences of repeated cold exposure differ from the impacts of a single cold exposure. We tested the hypothesis that different kinds of cold exposure (in this case, single short, prolonged and repeated cold exposure) would result in differential gene expression. We exposed 3 day old adult female wild-type Drosophila melanogaster (Diptera: Drosophilidae) to -0.5°C for a single 2 h exposure, a single 10 h exposure, or five 2 h exposures on consecutive days, and extracted RNA after 6 h of recovery. Global gene expression was quantified using an oligonucleotide microarray and validated with real-time PCR using different biological replicates. We identified 76 genes upregulated in response to multiple cold exposure, 69 in response to prolonged cold exposure and 20 genes upregulated in response to a single short cold exposure, with a small amount of overlap between treatments. Three genes--Turandot A, Hephaestus and CG11374--were upregulated in response to all three cold exposure treatments. Key functional groups upregulated include genes associated with muscle structure and function, the immune response, stress response, carbohydrate metabolism and egg production. We conclude that cold exposure has wide-ranging effects on gene expression in D. melanogaster and that increased duration or frequency of cold exposure has impacts different to those of a single short cold exposure. This has important implications for extrapolating laboratory studies of insect overwintering that are based on only a single cold exposure.

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster.

BACKGROUND: The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in Drosophila different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in Drosophila Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response. RESULTS: Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3rd instar larvae and we show that 20E-induced levels of EcR isoforms EcR-RA, ER-RC, and EcR-RD/E differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types. CONCLUSIONS: We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.

Identification of heat shock factor 1 molecular and cellular targets during embryonic and adult female meiosis.

Heat shock factor 1 (HSF1), while recognized as the major regulator of the heat shock transcriptional response, also exerts important functions during mammalian embryonic development and gametogenesis. In particular, HSF1 is required for oocyte maturation, the adult phase of meiosis preceding fertilization. To identify HSF1 target genes implicated in this process, comparative transcriptomic analyses were performed with wild-type and HSF-deficient oocytes. This revealed a network of meiotic genes involved in cohesin and synaptonemal complex (SC) structures, DNA recombination, and the spindle assembly checkpoint (SAC). All of them were found to be regulated by HSF1 not only during adult but also in embryonic phases of female meiosis. Additional investigations showed that SC, recombination nodules, and DNA repair were affected in Hsf1(-/-) oocytes during prenatal meiotic prophase I. However, targeting Hsf1 deletion to postnatal oocytes (using Zp3 Cre; Hsf1(loxP/loxP)) did not fully rescue the chromosomal anomalies identified during meiotic maturation, which possibly caused a persistent SAC activation. This would explain the metaphase I arrest previously described in HSF1-deficient oocytes since SAC inhibition circumvented this block. This work provides new insights into meiotic gene regulation and points out potential links between cellular stress and the meiotic anomalies frequently observed in humans.

Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster.

During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions.

Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.

The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the "writers" of the neuronal epigenome and how they lay down the basis for proper cognition. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, we generated genome-wide H3K9 dimethylation profiles by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5' and 3' ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. Our study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease.

Chronic alcohol exposure induced gene expression changes in the zebrafish brain.

Chronic alcohol exposure affects the central nervous system, influences behavior, and induces neuroadaptive changes in vertebrate species including our own. The molecular mechanisms responsible for chronic alcohol effects have not been fully elucidated due to the complexity of alcohol's actions. Here we use zebrafish, a novel tool in alcohol research, to reveal a large number of genes that respond to chronic alcohol treatment. We demonstrate differential gene expression in response to chronic alcohol treatment using full genome DNA microarrays and find a total of 1914 genes to show a minimum of 2-fold and significant expression level change (1127 were up- and 787 were down-regulated). Approximately two-thirds of these genes had no known previous functional annotation. The results of the microarray analyses correlated well with those obtained on a selected subset of genes analyzed by quantitative real-time RT-PCR. Analyses of the differentially expressed genes with known annotations were enriched for a variety of molecular functions. Only a fraction of these known genes has been reported in the literature to be alcohol related. We conclude that the zebrafish is an excellent tool for the analysis of genes associated with alcohol's actions in vertebrates, one which may facilitate the discovery and better understanding of the mechanisms of alcohol abuse.

Microarray analysis of gene expression by skeletal muscle of three mouse models of Kennedy disease/spinal bulbar muscular atrophy.

BACKGROUND: Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR. METHODOLOGY/PRINCIPAL FINDINGS: We performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntington's Disease, and to those common to muscle atrophy from diverse causes. CONCLUSIONS/SIGNIFICANCE: By comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.

Gene expression profiling implicates OXPHOS complexes in lifespan extension of flies over-expressing a small mitochondrial chaperone, Hsp22.

Aging is a complex process accompanied by a decreased capacity to tolerate and respond to various stresses. Heat shock proteins as part of cell defense mechanisms are up-regulated following stress. In Drosophila, the mitochondrial Hsp22 is preferentially up-regulated in aged flies. Its over-expression results in an extension of lifespan and an increased resistance to stress. Hsp22 has chaperone-like activity in vitro, but the mechanism(s) by which it increases lifespan in flies are unknown. Genome-wide analysis was performed on long-lived Hsp22+ and control flies to unveil transcriptional changes brought by Hsp22. Transcriptomes obtained at 45days, 90% and 50% survival were then compared between them to focus more on genes up- or down-regulated in presence of higher levels of hsp22 mRNA. Hsp22+ flies display an up-regulation of genes mainly related to mitochondrial energy production and protein biosynthesis, two functions normally down-regulated during aging. Interestingly, among the 26 genes up-regulated in Hsp22+ flies, 7 genes encode for mitochondrial proteins, 5 of which being involved in OXPHOS complexes. Other genes that could influence aging such as CG5002, dGCC185 and GstS1 also displayed a regulation linked to Hsp22 expression. The up-regulation of genes of the OXPHOS system in Hsp22+ flies suggest that mitochondrial homeostasis is at the center of Hsp22 beneficial effects on lifespan.

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

Measuring mRNA stability during early Drosophila embryogenesis.

Maternal mRNAs play a major role in directing early Drosophila melanogaster development, and thus, precise posttranscriptional regulation of these messages is imperative for normal embryogenesis. Although initially abundant on egg deposition, a subset of these maternal mRNAs is targeted for destruction during the first 2 to 3 h of embryogenesis. In this chapter, we describe molecular methods to determine the kinetics and mechanisms of maternal mRNA decay in the early D. melanogaster embryo. We show how both unfertilized eggs and fertilized embryos can be used to identify maternal mRNAs destined for degradation, to explain changes in decay kinetics over time, and to uncover the molecular mechanisms of targeted maternal mRNA turnover. In the first section, we explore the methods and outcomes of measuring decay on a "gene-by-gene" basis, which involves examination of a small number of transcripts by Northern blotting, RNA dot blotting, and real-time RT-PCR. In the second section, we provide a comprehensive examination of the applications of microarray technology to study global changes in maternal mRNA decay during early development. Genome-wide surveys of maternal mRNA turnover provide a wealth of information regarding the magnitude, temporal regulation, and genetic control of maternal mRNA turnover. Methods that permit the collection and analysis of highly reproducible and statistically robust data in this developmental system are discussed.