Project description:To evaluate the specificity of long dsRNAs used in high-throughput RNAi screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Surprisingly, our analysis predicts that dsRNAs containing ≥19 nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to the efficient knockdown of a cross-hybridizing transcript, raising a cautionary note when interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high quality information from RNAi high-throughput screens Keywords: Specificity of long dsRNAs, Drosophila melanogaster, SL2 cell line, custom cDNA arrays, off-target effects, Drosophila RNAi Screening Center (DRSC)
Project description:It has long been appreciated that striped pair-rule transcription factor expression is necessary for convergent extension in the early Drosophila embryo, although the mechanisms that link these transcriptional regulators to planar polarity in this tissue have long been elusive. The goal of this study was to determine the transcriptional tragets of the pair-rule transcription factors Eve and Runt in Drosophila blastoderm embryos. We compared the transcriptional profiles of late blastoderm embryos injected with either water or dsRNAs against both eve and runt to identify differentially expressed genes that may directly contribute to the establishment of planar polarity during Drosophila convergent extension. Comparing the mRNA profiles from late blastoderm Drosophila embryos injected with either water (Water) or eve+runt dsRNAs (Eve), in triplicate, using Illumina HiSeq.
Project description:To verify unannotated translated open reading frames (utORFs) identified from Drosophila melanogaster, we collected data to target them.
Project description:The notion that genes are the sole units of heredity and that a barrier exists between soma and germline has been a major hurdle in elucidating the heritability of traits that were observed to follow a non-Mendelian inheritance pattern. It was only after the conception of “epigenetics” by C. H. Waddington that the effect of parental environment on subsequent generations via non-DNA sequence-based mechanisms, such as DNA methylation, chromatin modifications, non-coding RNAs and proteins, could be established in various organisms, now referred to as multigenerational epigenetic inheritance. Despite the growing body of evidence, the male gamete-derived epigenetic factors that mediate the transmission of such phenotypes are seldom explored, particularly in the model organism Drosophila melanogaster. Using the heat stress-induced multigenerational epigenetic inheritance paradigm in a widely used position-effect variegation line of Drosophila, named white-mottled, we have dissected the effect of heat stress on the sperm proteome in the current study. We demonstrate that multiple successive generations of heat stress at the early embryonic stage results in a significant downregulation of proteins associated with translation, chromatin organization, microtubule-based processes, and generation of metabolites and energy in the Drosophila sperms. Based on our findings, we propose chromatin-based epigenetic mechanisms, a well-established mechanism for environmentally induced multigenerational effects, as a plausible way of transmitting heat stress memory via the male germ line in subsequent generations. Moreover, we demonstrate the effect of multiple generations of heat stress on the reproductive fitness of Drosophila, shedding light on the adaptive or maladaptive potential of heat stress-induced multigenerational phenotypes.