RESUMO
BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de Inseto , Janus Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição/genética , Animais , Células Cultivadas , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Janus Quinases/metabolismo , Luciferases , Plasmídeos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.
Assuntos
Perfilação da Expressão Gênica , Neoplasias Hematológicas/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Transcrição Gênica , Animais , Proliferação de Células , Análise por Conglomerados , Drosophila melanogaster , Regulação da Expressão Gênica , Células HeLa , Neoplasias Hematológicas/patologia , Hemócitos/citologia , Humanos , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3-GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance.