TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an 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.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Circadian neuron feedback controls the Drosophila sleep--activity profile.

Little is known about the ability of Drosophila circadian neurons to promote sleep. Here we show, using optogenetic manipulation and video recording, that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep. To investigate more plastic aspects of the sleep program, we used a calcium assay to monitor and compare the real-time activity of DN1 neurons in freely behaving males and females. Our results revealed that DN1 neurons were more active in males than in females, consistent with the finding that male flies sleep more during the day. DN1 activity is also enhanced by elevated temperature, consistent with the ability of higher temperatures to increase sleep. These new approaches indicate that DN1s have a major effect on the fly sleep-wake profile and integrate environmental information with the circadian molecular program.

RNA-seq analysis of Drosophila clock and non-clock neurons reveals neuron-specific cycling and novel candidate neuropeptides.

Locomotor activity rhythms are controlled by a network of ~150 circadian neurons within the adult Drosophila brain. They are subdivided based on their anatomical locations and properties. We profiled transcripts "around the clock" from three key groups of circadian neurons with different functions. We also profiled a non-circadian outgroup, dopaminergic (TH) neurons. They have cycling transcripts but fewer than clock neurons as well as low expression and poor cycling of clock gene transcripts. This suggests that TH neurons do not have a canonical circadian clock and that their gene expression cycling is driven by brain systemic cues. The three circadian groups are surprisingly diverse in their cycling transcripts and overall gene expression patterns, which include known and putative novel neuropeptides. Even the overall phase distributions of cycling transcripts are distinct, indicating that different regulatory principles govern transcript oscillations. This surprising cell-type diversity parallels the functional heterogeneity of the different neurons.

CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in Drosophila.

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity. Previous work indicates that repression is due to a direct PER-CLK/CYC interaction as well as CLK/CYC phosphorylation. We describe here the role of ubiquitin-specific protease 8 (USP8) in circadian transcriptional repression as well as the importance of CLK ubiquitylation in CLK/CYC transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances CLK/CYC transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, CLK ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal CLK/CYC transcription. As USP8 interacts with CLK and expression of USP8-DN increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK/CYC transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by CLK/CYC, USP8, like PER and TIM, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression.

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.

Longevity of centenarians is reflected by the gut microbiome with youth-associated signatures.

Centenarians are an excellent model to study the relationship between the gut microbiome and longevity. To characterize the gut microbiome signatures of aging, we conducted a cross-sectional investigation of 1,575 individuals (20-117 years) from Guangxi province of China, including 297 centenarians (n = 45 with longitudinal sampling). Compared to their old adult counterparts, centenarians displayed youth-associated features in the gut microbiome characterized by an over-representation of a Bacteroides-dominated enterotype, increase in species evenness, enrichment of potentially beneficial Bacteroidetes and depletion of potential pathobionts. Health status stratification in older individuals did not alter the directional trends for these signature comparisons but revealed more apparent associations in less healthy individuals. Importantly, longitudinal analysis of centenarians across a 1.5-year period indicated that the youth-associated gut microbial signatures were enhanced with regard to increased evenness, reduction in interindividual variation and stability of Bacteroides, and that centenarians with low microbial evenness were prone to large microbiome instability during aging. These results together highlight a youth-related aging pattern of the gut microbiome for long-lived individuals.

NonA and CPX Link the Circadian Clockwork to Locomotor Activity in Drosophila.

Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein PERIOD (PER) and contributes to circadian timekeeping. Here, we investigate NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor CLOCK/CYCLE (CLK/CYC), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.

Gene-specific RNA polymerase II phosphorylation and the CTD code.

Phosphorylation of the RNA polymerase (Pol) II C-terminal domain (CTD) repeats (1-YSPTSPS-7) 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 Ser2, Ser5 and Ser7 phosphorylations and the CTD-associated termination factors Nrd1 and Pcf11. Phospho-CTD dynamics are not scaled to gene length and are gene-specific, with highest Ser5 and Ser7 phosphorylation at the 5' ends of well-expressed genes with nucleosome-occupied promoters. The CTD kinases Kin28 and Ctk1 markedly affect Pol II distribution in a gene-specific way. The code is therefore written differently on different genes, probably under the control of promoters. Ser7 phosphorylation is enriched on introns and at sites of Nrd1 accumulation, suggesting links to splicing and Nrd1 recruitment. Nrd1 and Pcf11 frequently colocalize, suggesting functional overlap. Unexpectedly, Pcf11 is enriched at centromeres and Pol III-transcribed genes.

The role of Rat1 in coupling mRNA 3'-end processing to transcription termination: implications for a unified allosteric-torpedo model.

The torpedo model of transcription termination by RNA polymerase II proposes that a 5'-3' RNA exonuclease enters at the poly(A) cleavage site, degrades the nascent RNA, and eventually displaces polymerase from the DNA. Cotranscriptional degradation of nascent RNA has not been directly demonstrated, however. Here we report that two exonucleases, Rat1 and Xrn1, both contribute to cotranscriptional degradation of nascent RNA, but this degradation is not sufficient to cause polymerase release. Unexpectedly, Rat1 functions in both 3'-end processing and termination by enhancing recruitment of 3'-end processing factors, including Pcf11 and Rna15. In addition, the cleavage factor Pcf11 reciprocally aids in recruitment of Rat1 to the elongation complex. Our results suggest a unified allosteric/torpedo model in which Rat1 is not a dedicated termination factor, but is an integrated component of the cleavage/polyadenylation apparatus.

A ribonucleolytic rat torpedoes RNA polymerase II.

Three recent papers reveal a fascinating link between pol II termination and ribonucleolytic decay of the nascent transcript by a 5'-3' exonuclease (yeast Rat1 and human Xrn2). The exonuclease travels with pol II and gains access to the nascent RNA after endonucleolytic cleavage at the poly(A) site or at a second cotranscriptional cleavage site (CoTC). It is then thought to track in a 5'-3' direction like a guided torpedo that ultimately helps dissociate the RNA polymerase elongation complex.