LNd, LNv, DN1 and TH neurons were manually sorted from dissociated Drosophila brains. RNA was extracted and transcriptomes analyzed via RNA-seq. Overall design: LNd, LNv, DN1 and TH neurons at various timepoints.
Striking circadian neuron diversity and cycling of <i>Drosophila</i> alternative splicing.
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Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrated a requirement of neuronal SOCE for Drosophila flight. We identified the early pupal stage to be critical and used RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervous system. We down-regulated dStim, the endoplasmic reticular calcium sensor and a principal component of SOCE in the nervous system for a 24h period during pupal development, and compared wild type and knockdown transcriptional profiles, immediately after knockdown as well as after a 36h recovery period. We found that dStim knockdown altered the expression of a number of genes. We also characterized one of the down-regulated genes, Ral for its role in flight. Thus, we identify neuronal SOCE as a mechanism that regulates expression of a number of genes during the development of the pupal nervous system. These genes can be further studied in the context of pupal nervous system development. Overall design: mRNA sequencing from two biological replicates each of wild type and dStim knockdown pupal brains at two time points - 36h APF (post 24h knockdown) and at 72h APF (Post knockdown and recovery)
A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons.
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To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF (pigment-dispersing factor)-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv-enriched mRNA population including core clock mRNAs is shared between the l-LNvs and s-LNvs, the other half is l-LNv- and s-LNv specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well-expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons.
Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila.
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To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. 87% of introns assayed manifest more than 50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly, or slowly, with ~3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns and introns annotated as alternative. FinallyFinally, S2 cells expressing the slow RpII215C4 mutant manifest substantially less intron retention than wild-type S2 cells. Overall design: Examination of Total pA and Nascent RNA from 2 different cell populations and isolated fly heads.
Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila.
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