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Isolation of mRNA from fly photoreceptor cells by mRNA tagging

ABSTRACT: The method of mRNA tagging was recently developed to isolate mRNA population from muscle tissues in Caenohabditis elegans. This method utilizes a FLAG-tagged poly(A)-binding protein (PABP), which can bind to poly(A)-tail of eukaryotic mRNA. Thus, the mRNA population from specific tissue, in which FLAG- tagged PABP is expressed, can be co-immunoprecipitated with FLAG-tagged PABP using FLAG-specific antibody. To evaluate the applicability of this method to isolate mRNA from specific tissue in Drosophila, we generated Rh1-GLA4/P{UAS-dPABP-FLAG} flies which express a FLAG-tagged PABP in photoreceptor cells R1-R6. We then prepared 4 individual mRNA samples (subset 1: GSM30900-30903)) presumably from photoreceptor cells of these flies by mRNA tagging method. We also prepared 4 individual mRNA samples (subset 2: GSM30832-30835) from whole heads of the Rh1-GAL4/P{UAS-dPABP-FLAG} flies, as well as 6 individual mRNA samples (subset 3: GSM30824-30828, GSM30830) from whole heads of wild type Canton-S flies. The total 14 mRNA samples were used to synthesize target separately and each of the resultant target was used to hybridize with 1 Drosophila Genome Array DrosGenome1 Affymetrix GeneChip. The raw data of the 14 microarray were imported to dChip program (version 1.3). The arrays were normalized to a common baseline array that has the median overall brightness. The expression value for each probe set on each chip was then calculated as model-based expression index (MBEI) by Perfect Match-only model. By comparing the results of subset 1 and subset 2, we found most known photoreceptor-specific genes were indeed enriched by mRNA tagging. Through the comparison of the results of subset 2 and subset 3, we found the mRNAs of most genes known to be involved in fly visual function were underrepresented in subset 2, which could be due to squelching effect of GAL4. By combining the data of subset 1, 2 and 3, we were able to identify 11 new photoreceptor cell-enriched genes. Keywords: other

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE1790 | GEO | 2004/10/11

SECONDARY ACCESSION(S): PRJNA90587

REPOSITORIES: GEO

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Project description:The method of mRNA tagging was recently developed to isolate mRNA population from muscle tissues in Caenohabditis elegans. This method utilizes a FLAG-tagged poly(A)-binding protein (PABP), which can bind to poly(A)-tail of eukaryotic mRNA. Thus, the mRNA population from specific tissue, in which FLAG- tagged PABP is expressed, can be co-immunoprecipitated with FLAG-tagged PABP using FLAG-specific antibody. To evaluate the applicability of this method to isolate mRNA from specific tissue in Drosophila, we generated Rh1-GLA4/P{UAS-dPABP-FLAG} flies which express a FLAG-tagged PABP in photoreceptor cells R1-R6. We then prepared 4 individual mRNA samples (subset 1: GSM30900-30903)) presumably from photoreceptor cells of these flies by mRNA tagging method. We also prepared 4 individual mRNA samples (subset 2: GSM30832-30835) from whole heads of the Rh1-GAL4/P{UAS-dPABP-FLAG} flies, as well as 6 individual mRNA samples (subset 3: GSM30824-30828, GSM30830) from whole heads of wild type Canton-S flies. The total 14 mRNA samples were used to synthesize target separately and each of the resultant target was used to hybridize with 1 Drosophila Genome Array DrosGenome1 Affymetrix GeneChip. The raw data of the 14 microarray were imported to dChip program (version 1.3). The arrays were normalized to a common baseline array that has the median overall brightness. The expression value for each probe set on each chip was then calculated as model-based expression index (MBEI) by Perfect Match-only model. By comparing the results of subset 1 and subset 2, we found most known photoreceptor-specific genes were indeed enriched by mRNA tagging. Through the comparison of the results of subset 2 and subset 3, we found the mRNAs of most genes known to be involved in fly visual function were underrepresented in subset 2, which could be due to squelching effect of GAL4. By combining the data of subset 1, 2 and 3, we were able to identify 11 new photoreceptor cell-enriched genes.

Project description:To investigate the in vivo gene expression changes in Drosophila intestinal stem cells (ISCs) when OXPHOS is disrupted and the role of FOXO in regulating this process, we dissected midguts from 4 groups of adult flies: "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & homoplasmic mt:CoIsyn" (C group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & heteroplasmic mt:CoIT300I" (H group, also called M group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & homoplasmic mt:CoIsyn" (FC group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & heteroplasmic mt:CoIT300I" (FH group, also called FM group), ~200 midguts per group. The flies were initially obtained and maintained at 18 °C till 2 days after eclosion and were then heat-shocked at 37 ºC for 2 hours and maintained in 29 °C for 5 days. Thus, the ISCs of H and FH group flies were carrying the lethal homoplasmic mitoXhoI-resistant mt:CoIT300I mitochondrial DNAs (mtDNAs) with the wild-type mtDNAs eliminated by mitoXhoI and the OXPHOS disrupted. Meanwhile, the ISCs of C and FC group flies were still carrying wild-type-like but mitoXhoI-resistant mt:CoIsyn mtDNAs. Through the mRNA tagging method with the expressed FLAG-tagged poly(A)-binding protein (pAbp) and anti-FLAG affinity pull-down, We purified and compared mRNA transcriptome profiles (RNA-seq) of different ISCs groups in duplicates. We found about 10% of the mRNAs we detected were either up-regulated or down-regulated in the OXPHOS-disrupted (M group) ISCs compared with control (C group). The down-regulated pool included many genes involved in cell differentiation, proliferation, and growth regulation. The up-regulated pool was enriched in genes participating in various metabolic processes, including glycolysis and fatty acids synthesis. This result suggests a possible metabolic adaptation in response to the OXPHOS deficiency in M group ISCs. Importantly, knockdown of FOXO in OXPHOS-disrupted ISCs restored the expression level of more than 50% of the up- or down-regulated genes, indicating that transcriptional response to OXPHOS deficiency is partially mediated by FOXO. We also noticed that genes whose expressions were altered in OXPHOS-disrupted ISCs included many related to Notch signaling, indicating the mis-regulation of this signaling in these ISCs.

Project description:Background: With its fully sequenced genome and simple, well-defined nervous system, the nematode C. elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and thereby link comprehensive profiles of neuronal transcription to key developmental and functional attributes of the nervous system. Results: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Micro-Array Profiling C. elegans Cells) method, we used Fluorescence Activated Cell Sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (e.g. transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. In addition to utilizing these profiling approaches to define stage specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. Conclusion: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and therefore provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system. Experiment Overall Design: Our goal is to profile gene expression throughout the nervous system of the model organism Caenorhabditis elegans. As a first goal, we profiled the entire nervous system at two developmental timepoints. To isolate transcripts from embryonic neurons we employed the MAPCeL (Microarray Profiling C. elegans Cells) technique in which pan-neuronal GFP+ cells are captured by FACS for RNA isolation. Since postembryonic cells are unattainable via this method, we used mRNA-tagging to isolated pan-neural RNAs from larval animals. In short, an epitope tagged poly-A binding protein (FLAG-PAB-1) was driven in all neurons using the F25B3.3 promoter. Following formaldehyde cross-linking, animals were passed through a French press and Dounce homogenized to isolate a cell-free extract. Anti-FLAG antibodies were incubated with the cell-free extract to capture FLAG-PAB bound RNAs. After elution and reversal of the crosslinks, RNAs are isolated by TriZOL extraction. We also identified transcripts enriched in a subset of C. elegans larval neurons, the A-class neurons (using unc-4::3xFLAG::PAB-1). Experiment Overall Design: 3 sets of experiments normalized separately by RMA Experiment Overall Design: larval references of sets 2 and 3 originate from the same hybridizations (accompanying CEL files are identical)

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Isolation of mRNA from fly photoreceptor cells by mRNA tagging

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