SUMMARY Little is known about the ability of Drosophila circadian neurons to promote sleep. We show here with optogenetic manipulations and video recording that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells, releasing glutamate to directly inhibit key pacemaker neurons. These pacemakers promote morning arousal by activating these same DN1s, implying that there is a late-day feedback circuit to drive siesta and nighttime sleep. To address more plastic aspects of the sleep program, we used a novel calcium assay to monitor and compared the real-time DN1 activity of freely behaving males and females. It revealed a dramatic sexual dimorphism, which parallels the well-known difference in daytime sleep. DN1 activity is also enhanced by elevated temperature, consistent with its known effect on sleep. These new approaches indicate that the DN1s have a major impact on the fly sleep-wake profile and integrate environmental information with the circadian molecular program.
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of a RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (Targets of RNA-binding proteins Identified By Editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1 and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.
Dynamic phosphorylation of the RNA polymerase II CTD repeats (YS2PTS5PS7) is coupled to transcription and may act as a “code” that controls mRNA synthesis and processing. To examine the "code" in budding yeast, we mapped genome-wide CTD S2, 5 and 7 phosphorylations (PO4) and compared them with the CTD-associated termination factors, Nrd1 and Pcf11. CTD-PO4 dynamics are not scaled to the size of the gene. At 5’ ends, the onset of S2-PO4 is delayed by about 450 bases relative to S5-PO4, regardless of gene length. Phospho-CTD dynamics are gene-specific, with high S5/7-PO4 at the 5' end being characteristic of well-expressed genes with nucleosome-occupied promoters. Furthermore, the CTD kinases Kin28 and Ctk1 profoundly affect pol II distribution along genes in a highly gene-specific way. The "code" is therefore written differently on different genes, probably under the control of promoters. S7-PO4 is enriched on introns and at sites of Nrd1 accumulation suggesting that this modification may function in splicing and Nrd1 recruitment. Nrd1 and Pcf11 frequently co-localized, suggesting functional overlap between these terminators. Surprisingly, Pcf11 is also recruited to centromeres and pol III transcribed genes.
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