Person: Mohr, Stephanie
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Mohr
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Stephanie
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Mohr, Stephanie
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Publication Loss-of-Function Genetic Tools for Animal Models: Cross-Species and Cross-Platform Differences(Springer Science and Business Media LLC, 2017-01) Housden, Benjamin; Muhar, Matthias; Gemberling, Matthew; Gersbach, Charles A.; Stainier, Didier Y.R.; Seydoux, Geraldine; Mohr, Stephanie; Zuber, Johannes; Perrimon, NorbertOur understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.Publication RNAi Screening Comes of Age: Improved Techniques and Complementary Approaches(Springer Science and Business Media LLC, 2014-09) Mohr, Stephanie; Smith, Jennifer; Shamu, Caroline; Neumüller, Ralph A.; Perrimon, NorbertGene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.Publication Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data(Oxford University Press, 2018) Hu, Yanhui; Vinayagam, Arunachalam; Nand, Ankita; Comjean, Aram; Chung, Verena; Hao, Tong; Mohr, Stephanie; Perrimon, NorbertAbstract Model organism and human databases are rich with information about genetic and physical interactions. These data can be used to interpret and guide the analysis of results from new studies and develop new hypotheses. Here, we report the development of the Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/). The MIST database integrates biological interaction data from yeast, nematode, fly, zebrafish, frog, rat and mouse model systems, as well as human. For individual or short gene lists, the MIST user interface can be used to identify interacting partners based on protein–protein and genetic interaction (GI) data from the species of interest as well as inferred interactions, known as interologs, and to view a corresponding network. The data, interologs and search tools at MIST are also useful for analyzing ‘omics datasets. In addition to describing the integrated database, we also demonstrate how MIST can be used to identify an appropriate cut-off value that balances false positive and negative discovery, and present use-cases for additional types of analysis. Altogether, the MIST database and search tools support visualization and navigation of existing protein and GI data, as well as comparison of new and existing data.Publication Gene2Function: An Integrated Online Resource for Gene Function Discovery(Genetics Society of America, 2017) Hu, Yanhui; Comjean, Aram; Mohr, Stephanie; Perrimon, NorbertOne of the most powerful ways to develop hypotheses regarding the biological functions of conserved genes in a given species, such as humans, is to first look at what is known about their function in another species. Model organism databases and other resources are rich with functional information but difficult to mine. Gene2Function addresses a broad need by integrating information about conserved genes in a single online resource.Publication FlyPrimerBank: An Online Database for Drosophila melanogaster Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents(Genetics Society of America, 2013) Hu, Yanhui; Sopko, Richelle; Foos, Marianna; Kelley, Colleen; Flockhart, Ian; Ammeux, Noemie; Wang, Xiaowei; Perkins, Lizabeth A.; Perrimon, Norbert; Mohr, StephanieThe evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.Publication Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis(BioMed Central, 2014) Yilmazel, Bahar; Hu, Yanhui; Sigoillot, Frederic; Smith, Jennifer A; Shamu, Caroline; Perrimon, Norbert; Mohr, StephanieBackground: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. Results: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files. Conclusion: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences.Publication Integrating protein-protein interaction networks with phenotypes reveals signs of interactions(2013) Vinayagam, Arunachalam; Zirin, Jonathan; Roesel, Charles; Hu, Yanhui; Yilmazel, Bahar; Samsonova, Anastasia A.; Neumüller, Ralph A.; Mohr, Stephanie; Perrimon, NorbertA major objective of systems biology is to organize molecular interactions as networks and to characterize information-flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the “signs” of interactions (i.e. activation/inhibition relationships). We constructed a Drosophila melanogaster signed PPI network, consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes Enolase and Aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation/inhibition relationships between physically interacting proteins within signaling pathways will impact our understanding of many biological functions, including signal transduction and mechanisms of disease.Publication Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells(Genetics Society of America, 2015) Mohr, Stephanie; Hu, Yanhui; Rudd, Kirstin; Buckner, Michael; Gilly, Quentin; Foster, Blake; Sierzputowska, Katarzyna; Comjean, Aram; Ye, Bing; Perrimon, NorbertRNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.Publication FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update(Oxford University Press, 2017) Hu, Yanhui; Comjean, Aram; Roesel, Charles; Vinayagam, Arunachalam; Flockhart, Ian; Zirin, Jonathan; Perkins, Lizabeth A.; Perrimon, Norbert; Mohr, StephanieThe FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.Publication GLAD: an Online Database of Gene List Annotation for Drosophila(Ivyspring International Publisher, 2015) Hu, Yanhui; Comjean, Aram; Perkins, Lizabeth A.; Perrimon, Norbert; Mohr, StephanieWe present a resource of high quality lists of functionally related Drosophila genes, e.g. based on protein domains (kinases, transcription factors, etc.) or cellular function (e.g. autophagy, signal transduction). To establish these lists, we relied on different inputs, including curation from databases or the literature and mapping from other species. Moreover, as an added curation and quality control step, we asked experts in relevant fields to review many of the lists. The resource is available online for scientists to search and view, and is editable based on community input. Annotation of gene groups is an ongoing effort and scientific need will typically drive decisions regarding which gene lists to pursue. We anticipate that the number of lists will increase over time; that the composition of some lists will grow and/or change over time as new information becomes available; and that the lists will benefit the scientific community, e.g. at experimental design and data analysis stages. Based on this, we present an easily updatable online database, available at www.flyrnai.org/glad, at which gene group lists can be viewed, searched and downloaded.
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