Locomotor activity rhythms are controlled by a network of ~150 circadian neurons within the adult Drosophila brain. They subdivided based on their anatomical locations and properties. We profiled transcripts "around clock" from three key groups with different functions. also non-circadian outgroup, dopaminergic (TH) neurons. have cycling but fewer than clock as well low expression poor gene transcripts. This suggests that TH do not canonical is driven brain systemic cues. The surprisingly...

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity cell lines fly strains, even for identically named sub-strains from different laboratories such as ISO1 strain used reference genome sequence. On average, >500 novel insertions exist every lab strain, inbred strains Genetic...

The Piwi pathway is deeply conserved amongst animals because one of its essential functions to repress transposons. However, many Piwi-interacting RNAs (piRNAs) do not base-pair transposons and remain mysterious in their targeting function. sheer number piRNA cluster (piC) loci animal genomes infrequent sequence conservation also present challenges determining which piC are most important for development. To address this question, we determined the expression patterns across a wide...

Activity-regulated genes (ARGs) are important for neuronal functions like long-term memory and well-characterized in mammals but poorly studied other model organisms Drosophila. Here we stimulated fly neurons with different paradigms identified ARGs using high-throughput sequencing from brains as well sorted neurons: they included a narrow set of circadian dopaminergic neurons. Surprisingly, many specific to the stimulation paradigm very neuron type. In addition unlike mammalian immediate...

We previously developed TRIBE, a method for the identification of cell-specific RNA-binding protein targets. TRIBE expresses an RBP interest fused to catalytic domain (cd) RNA-editing enzyme ADAR and performs adenosine-to-inosine editing on RNA targets RBP. However, target is limited by low efficiency ADARcd. Here we describe HyperTRIBE, which carries characterized hyperactive mutation (E488Q) HyperTRIBE identifies dramatically more sites, many are also edited but at much lower frequency....

Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression aged animals. Does aging lead to more genomic copies older animals? To address this question, we quantified Landscapes (TLs) via whole genome sequencing of and Drosophila strains wild-type mutant backgrounds. We TLs flies dissected brains validated the feasibility our approach detecting new insertions genomes when small RNA interference (RNAi) pathways...

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, we conducted a genome-wide survey for transcripts associated with PIWI affected by knockdown Drosophila ovarian somatic sheet (OSS) cells, follicle cell line expressing pathway. Despite immense sequence diversity among OSS piRNAs, our analysis indicates TE are major directly regulated PIWI....

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. is an RNA-binding protein which forms aggregates neurons of ALS FTD patients as well a subset diagnosed other diseases. Despite our understanding essential for many aspects RNA metabolism, it remains obscure how dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology common...

4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although has been shown be important in growth control, stress response, cancer, neuronal activity, mammalian circadian rhythms, it is not understood how preferentially a subset of mRNAs. We successfully used HyperTRIBE (targets RNA proteins identified editing) identify vivo mRNA targets both Drosophila mammals under conditions known activate 4E-BP. The associates...

Both UV-cross-linking and immunoprecipitation (CLIP) RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives CLIP TRIBE, endogenous β-actin mRNA was tagged with MS2 stem loops, making it only bona fide target for capsid protein (MCP). TRIBE detected β-actin, albeit false-positives. False-positive signals were attributed to nonspecific antibody interactions. In contrast, putative false-positive genes spatially proximal gene. MCP-ADAR edited nearby...

Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome in Drosophila called gonadal dysgenesis (GD). Harwich (Har) strains with P-elements cause severe GD F1 daughters when Har fathers mate mothers lacking P-element-piRNAs (i.e. ISO1 strain). To address the mystery of why induces GD, we bred hybrid genomic fragments into background to create HISR-D or HISR-N lines that still Dysgenesis are Non-dysgenic, respectively. In these lines,...

Cuticular protein genes are good models to study the molecular mechanisms of signaling by ecdysteroids, which regulate molting and metamorphosis in insects. The present research demonstrates on hormonal regulation analysis regulatory sequences transcription factors important for Bombyx mori cuticular glycine-rich13 ( CPG13 ) gene expression. Expression was strong at prepupal stage wing tissues B. . expression induced addition 20E, inhibited cycloheximide disc. upstream region analyzed using...

The close relationship between RNA structure and function underlines the significance of accurately predicting structures from sequence information. Structural topologies such as pseudoknots are particular interest due to their ubiquity direct involvement in function, but identifying is a computationally challenging problem existing heuristic approaches usually perform poorly for sequences even few hundred bases. We survey performance pseudoknot prediction methods on data set full-length...

Abstract Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo crosslinking (“CLIP” and its variants), which rely on protein-RNA antibodies. Another recently introduced exploits editing, with the catalytic domain ADAR covalently attached a (“TRIBE”). Both approaches suffer from difficulties distinguishing real targets false negative especially positive signals. To critically evaluate this...

Abstract We previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP interest fused to catalytic domain (cd) editing enzyme ADAR and performs Adenosine-to-Inosine on targets RBP. However, target is limited by low efficiency ADARcd. Here we describe HyperTRIBE, which carries characterized hyperactive mutation (E488Q) HyperTRIBE identifies dramatically more sites, many are also edited but at much lower frequency. therefore...

The insect cuticle is composed of various proteins and formed during the moult under a complex biological process that depends on cross talk between hormone levels gene expression. In present study, we aimed to clarify ecdysone-dependent temporal regulation mechanisms cuticular expression underlying control Bombyx mori metamorphosis. CPR55 was observed from W3 early stage peaked at pupation when ecdysteroid titre declined. induced by ecdysone pulse, their 24 h after transfer free medium....

Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. is an RNA-binding protein forms aggregates neurons of ALS FTD patients as well a subset diagnosed other diseases. Despite our understanding essential for many aspects RNA metabolism, it remains obscure how dysfunction contributes to neurodegeneration. Interestingly, several neurological disorders display...

Abstract 4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although has been shown be important in growth control, stress response, cancer, neuronal activity mammalian circadian rhythms, it is not understood how preferentially a subset of mRNAs. We successfully used hyperTRIBE (Targets RNA-binding proteins identified editing) identify vivo mRNA targets both Drosophila mammals under conditions known activate 4E-BP. The...

ABSTRACT Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression aged animals. Does aging lead to more genomic copies older animals? To answer this question, we quantified Landscapes (TLs) via whole genome sequencing of and Drosophila strains wild-type mutant backgrounds. We TLs flies dissected brains validated the feasibility our approach detecting new insertions genomes when natural defenses like RNA...

Targets of RNA-binding proteins discovered by editing (TRIBE) determines RNA-proteins interactions and nuclear organization with minimal false positives. We detail necessary steps for performing mammalian cell RBP-TRIBE to determine the targets MS2-TRIBE RNA-RNA within nucleus. Necessary a TRIBE experiment are detailed, starting plasmid/cell line generation, cellular transfection, RNA sequencing library preparation concluding bioinformatics analysis sites identification target RNAs. For...