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C. elegans: total mRNA vs. AWA-specifc mRNA

ABSTRACT: Transcriptional profiling of AWA neurons comparing total mRNA of C. elegans The aforementioned results provided us a clear indication that a GPCR expressed in AWA would likely be used for pheromone perception. To get a male AWA-specific expression profile, we adopted a poly-A-binding protein (PABP) mRNA pull-down approach to isolate cell-specific genes (Kunitomo et al., 2005) and co-transformed into the worms a sex determination gene, fem-3, to masculinize its nervous system. Ectopic expression of fem-3 in all neurons can change the entire nervous system to adopt a male identity (Ahringer & Kimble, 1991; Rosenquist & Kimble, 1988). We established the male AWA single-cell transcriptional profile by pooling mRNA from AWA with ectopic expression of PABP there driven by an AWA-specific promoter from odr-7. This PABP fused with an epitope protein tag (FLAG tag) is then extracted using the corresponding antibody, while AWA enriched transcripts with poly-A tail, including the GPCRs, were pulled down together and subjected to microarray analysis. The results confirmed the existence of dyp-13 (hypodermal gene), myo-3 (muscle gene) and odr-10 (AWA-specific over-expression gene) in total RNA samples while odr-10 was found to be over-expressed in AWA enriched sample, where dpy-13 and myo-3 were depleted. The pull-down RNA sample was labeled with cyanine 5 (which is excited by a 532nm laser) and the total RNA sample was labeled with cyanine 3 (which is excited by a 633nm laser). Comparison between transcriptome profiles revealed key differences between whole worm and male AWA neurons, which allows identification of male AWA-enriched transcripts including those encoding GPCRs.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE112610 | GEO | 2018/04/03

REPOSITORIES: GEO

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Project description:To gain insight into the molecular changes of sleep need, this study addresses gene expression changes in a subpopulation of neurons selectively activated by sleep deprivation. Whole brain expression analyses after 6h sleep deprivation clearly indicate that Homer1a is the best index of sleep need, consistently in all mouse strains analyzed. Transgenic mice expressing a FLAG-tagged poly(A)-binding protein (PABP) under the control of Homer1a promoter were generated. Because PABP binds the poly(A) tails of mRNA, affinity purification of FLAG-tagged PABP proteins from whole brain lysates, is expected to co-precipitate all mRNAs from neurons expressing Homer1a. Three other activity-induced genes (Ptgs2, Jph3, and Nptx2) were identified by this technique to be over-expressed after sleep loss. All four genes play a role in recovery from glutamate-induced neuronal hyperactivity. The consistent activation of Homer1a suggests a role for sleep in intracellular calcium homeostasis for protecting and recovering from the neuronal activation imposed by wakefulness. Experiment Overall Design: Experiments were performed on male mice, 12 weeks of age +/- 1 week. Animals were housed in polycarbonate cages (31x18x18cm) in an experimental room with an ambient temperature varying from 23Â° to 25Â°C under a 12:12 hrs light/dark cycle. Food and water were available ad libitum. At light onset mice were either sleep deprived by gentle handling (n=10) or left undisturbed (n=10) for 6 hrs. Animals were then randomly sacrificed by cervical dislocation. Total RNA from the whole brain was isolated for control (n=4) and sleep deprived (n=4) using a commercial RNA extraction kit (RNeasy Lipid Tissue Kit, Quiagen). Specific Homer1a-expressing cells polyA RNAs were immunoprecipitated following the total brain crosslinking (1% formaldehyde perfusion) for sleep deprived (n=6) and control (n=6) animals. The total RNA from the pull-down supernatants were also harvested (n=4). To test for transcriptional changes after sleep deprivation Homer1a-expressing cells, we proceeded in 2 steps: (1) identify probe sets enriched in the pull-down extracts, (2) among those probe sets compare sleep deprivation to control condition in both pull-down (6 vs. 6 chip comparison) and whole-brain (4 vs. 4 chip comparison) extracts. 4728 probe sets were significantly enriched at 5% FDR when pull-downs were compared to both supernatant and whole-brain extracts.

Project description:Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3M-bM-^@M-^YUTR strongly demonstrate that interrogation of the 3M-bM-^@M-^YUTRome in specific tissues and cell types across development will greatly expand the identification of new 3M-bM-^@M-^YUTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3M-bM-^@M-^YUTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3M-bM-^@M-^YUTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3M-bM-^@M-^YUTRome will reveal a far greater diversity of novel 3M-bM-^@M-^YUTR isoform expression that is masked in the whole-worm 3M-bM-^@M-^YUTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3M-JM-< end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.

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C. elegans: total mRNA vs. AWA-specifc mRNA

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