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Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Background: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles., Results: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167)., Conclusion: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

The Non-Coding RNA (ncRNA) elements in the 3' Untranslated Regions (3'-UTRs) are known to participate in the genes' post-transcriptional regulations. Inferring co-expression patterns of the genes through clustering these 3'-UTR ncRNA elements will provide invaluable insights for studying their biological functions. In this paper, we propose an improved RNA structural clustering pipeline. Benchmark of the new pipeline on Rfam data demonstrates over 10% performance improvements compared to the traditional hierarchical clustering pipeline. By applying the new clustering pipeline to 3'-UTRs of Drosophila melanogaster's genome, we have successfully identified 184 ncRNA clusters with 91.3% accuracy. One of these clusters corresponds to genes that are preferentially expressed in male Drosophila. Another cluster contains genes that are responsible for the functions of septate junction in epithelial cells. These discoveries encourage more studies on novel post-transcriptional regulation mechanisms.

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.

Background: Germline stem cells (GSCs) are present in the gonads of Drosophila females and males, and their proper maintenance, as well as their correct differentiation, is essential for fertility and fecundity. The molecular characterization of factors involved in maintenance and differentiation is a major goal both in Drosophila and stem cell research. While genetic studies have identified many of these key factors, the use of genome-wide expression studies holds the potential to greatly increase our knowledge of these pathways., Results: Here we report a genome-wide expression study that uses laser cutting microdissection to isolate germline stem cells, somatic niche cells, and early differentiating germ cells from female and male gonads. Analysis of this data, in association with two previously published genome-wide GSC data sets, revealed sets of candidate genes as putatively expressed in specific cell populations. Investigation of one of these genes, CG10990 the Drosophila ortholog of mammalian Programmed cell death 4 (Pdcd4), reveals expression in female and male germline stem cells and early differentiating daughter cells. Functional analysis demonstrates that while it is not essential for oogenesis or spermatogenesis, it does function to promote the differentiation of GSCs in females. Furthermore, in females, Pdcd4 genetically interacts with the key differentiation gene bag of marbles (bam) and the stem cell renewal factor eIF4A, suggesting a possible pathway for its function in differentiation., Conclusions: We propose that Pdcd4 promotes the differentiation of GSC daughter cells by relieving the eIF4A-mediated inhibition of Bam.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Background: Discovering the functions of all genes is a central goal of contemporary biomedical research. Despite considerable effort, we are still far from achieving this goal in any metazoan organism. Collectively, the growing body of high-throughput functional genomics data provides evidence of gene function, but remains difficult to interpret., Results: We constructed the first network of functional relationships for Drosophila melanogaster by integrating most of the available, comprehensive sets of genetic interaction, protein-protein interaction, and microarray expression data. The complete integrated network covers 85% of the currently known genes, which we refined to a high confidence network that includes 20,000 functional relationships among 5,021 genes. An analysis of the network revealed a remarkable concordance with prior knowledge. Using the network, we were able to infer a set of high-confidence Gene Ontology biological process annotations on 483 of the roughly 5,000 previously unannotated genes. We also show that this approach is a means of inferring annotations on a class of genes that cannot be annotated based solely on sequence similarity. Lastly, we demonstrate the utility of the network through reanalyzing gene expression data to both discover clusters of coregulated genes and compile a list of candidate genes related to specific biological processes., Conclusions: Here we present the the first genome-wide functional gene network in D. melanogaster. The network enables the exploration, mining, and reanalysis of experimental data, as well as the interpretation of new data. The inferred annotations provide testable hypotheses of previously uncharacterized genes.

Sex chromosomes are primary determinants of sexual dimorphism in many organisms. These chromosomes are thought to arise via the divergence of an ancestral autosome pair and are almost certainly influenced by differing selection in males and females. Exploring how sex chromosomes differ from autosomes is highly amenable to genomic analysis. We examined global gene expression in Drosophila melanogaster and report a dramatic underrepresentation of X-chromosome genes showing high relative expression in males. Using comparative genomics, we find that these same X-chromosome genes are exceptionally poorly conserved in the mosquito Anopheles gambiae. These data indicate that the X chromosome is a disfavored location for genes selectively expressed in males.

Nonautonomous inductive signals from the soma and autonomous signals due to a 2X karyotype determine the sex of Drosophila melanogaster germ cells. These two signals have partially overlapping influences on downstream sex determination genes. The upstream OVO-B transcription factor is required for the viability of 2X germ cells, regardless of sexual identity, and for female germline sexual identity. The influence of inductive and autonomous signals on ovo expression has been controversial. We show that ovo-B is strongly expressed in the 2X germ cells in either a male or a female soma. This indicates that a 2X karyotype controls ovo-B expression in the absence of inductive signals from the female soma. However, we also show that female inductive signals positively regulate ovo-B transcription in the 1X germ cells that do not require ovo-B function. Genetic analysis clearly indicates that inductive signals from the soma are not required for ovo-B function in 2X germ cells. Thus, while somatic inductive signals and chromosome karyotype have overlapping regulatory influences, a 2X karyotype is a critical germline autonomous determinant of ovo-B function in the germline.

Identifies DNA sequences that lie within a genetic region known to include the stoned gene and that also reveals restriction site variations in two stoned lethal mutants in Drosophila melanogaster. Isolation and sequencing of cDNA and genomic clones; Translation of two proteins from the stoned locus in vivo.