Biology subjects

Yadlapalli, S.

Publications and source records attributed to Yadlapalli, S..

10 recordsLinked to original sources

bioRxiv2026

P-body sequestration of clock transcripts delays repressor synthesis to set circadian period in Drosophila

Negative-feedback oscillators require a delay between the accumulation of a repressor's mRNA and the action of its protein. In the circadian clock, this delay has been attributed largely to post-translational control of PERIOD (PER) stability and nuclear entry. The RNA-binding proteins shown to regulate per translation, ATAXIN2 and its partners, promote it, leaving open whether any step holds clock transcripts back before they are translated. Here, using time-resolved miniTurbo proximity labeling of endogenous PER across four phases of the circadian cycle in Drosophila clock neurons, we define a 252-protein PER proximitome that partitions into a nuclear arm and a cytoplasmic RNA-metabolism arm. A behavioral RNAi screen identified two P-body components, the DEAD-box helicase Me31B (DDX6) and the 5'-3' exonuclease Pacman (Pcm; XRN1), as strong regulators of circadian rhythms. Using single-molecule RNA-FISH, proximity RNA editing and ribosome profiling, we show that as per and tim transcripts accumulate, they localize to Me31B-labeled P-bodies and are poorly translated, most prominently at ZT12. Me31B knockdown disrupts P-bodies and releases per mRNA from them, causing PER to accumulate earlier and to ~2-fold higher levels, whereas Me31B overexpression delays PER accumulation and lengthens the free-running period by ~2 h. Knockdown of Pcm, in contrast, impairs clearance of per mRNA, sustaining PER and TIM accumulation, prolonging the repression phase and abolishing cycling of ~89% of rhythmic transcripts. Together, these findings identify P-body sequestration as a repressive step that delays repressor synthesis, and Pcm-dependent decay as required to end repression on time. Given the deep conservation of DDX6 and XRN1, RNP compartments may provide a conserved means of generating delay in circadian and other negative-feedback circuits.

cell biology↗

bioRxiv2026

Exposure to perfluorooctanoic acid accelerates Drosophila melanogaster juvenile development and disrupts mitochondrial metabolism

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with poorly understood sublethal effects on insects. Perfluorooctanoic acid (PFOA), one of the most widely distributed legacy PFAS is increasingly recognized for altering organismal physiology beyond traditional toxicity endpoints. Here, we use the fruit fly Drosophila melanogaster as a model to examine how PFOA exposure during larval (juvenile) development reshapes insect life-history progression and metabolic homeostasis. Our studies reveal that at environmentally relevant concentrations (nM to low {micro}M), PFOA induces precocious expression of developmentally-regulated genes and leads to metabolic changes that persist into adulthood. At higher concentrations used to probe mechanism, PFOA accelerates larval development, disrupts mitochondrial membrane potential, and increases whole-organism metabolic heat production - results that suggest altered mitochondrial energetic efficiency. Consistent with this tradeoff, PFOA-exposed larvae that develop faster under permissive conditions exhibit heightened sensitivity to environmental stressors, including elevated temperature and reduced food hydration. Together, these findings demonstrate that PFOA disrupts metabolic and developmental processes in a dose- and context-dependent manner, highlighting sublethal effects that may influence insect resilience under environmental stress. SYNOPSIS STATEMENTHere we describe how PFOA alters the growth, development, and metabolism of the fruit fly Drosophila melanogaster. Specifically, we find that PFOA accelerates Drosophila juvenile growth while also rendering exposed larvae sensitive to environmental stress. These observations suggest that widespread PFOA contamination may impair the developmental fitness of insect populations.

pharmacology and toxicology↗

bioRxiv2026

SWI/SNF nuclear foci scaffold peripheral gene hubs for circadian chromatin control

How dynamic circadian rhythms arise from a relatively static nuclear architecture remains a fundamental open question. Here, we identify the SWI/SNF chromatin-remodeling complex as a critical interface between the circadian clock and nuclear organization. Using endogenously tagged Moira (MOR), a core component of the Drosophila SWI/SNF complex and homolog of human BAF155, we find that MOR assembles into a small number of discrete foci near the nuclear periphery, in stark contrast to the current model of diffuse nuclear distribution for SWI/SNF-family proteins. We demonstrate that this localization is maintained by the inner nuclear envelope LEM-domain protein Otefin, effectively shifting MOR from a global to a localized regulator of chromatin architecture. DNA-FISH reveals that clock-regulated genes cluster into peripheral "hubs" that co-localize with MOR foci throughout the circadian cycle. ATAC-seq analysis shows that while MOR modulates chromatin accessibility genome-wide, it establishes a constitutive restrictive baseline specifically at clock-regulated loci. As a result, MOR depletion abolishes accessibility rhythms at these loci, rendering them constitutively hyper-accessible. This deregulation disrupts rhythmic gene expression and ultimately drives behavioral arrhythmia. Strikingly, oscillations of the core clock proteins PER and TIM remain intact, indicating that MOR loss uncouples the protein oscillator from its genomic output. Together, these results reveal that MOR-containing SWI/SNF foci form a stable perinuclear scaffold that gates chromatin accessibility, enabling the core clock machinery to convert transient protein oscillations into high-amplitude transcriptional rhythms. Significance StatementHow dynamic circadian rhythms emerge from a relatively static nuclear architecture remains a fundamental mystery. Here, we challenge the assumption that clock-target genes are randomly distributed by identifying a precise nuclear organization where these genes cluster into peripheral hubs. These hubs co-localize with stable SWI/SNF (MOR) foci anchored at the nuclear envelope. We demonstrate that this perinuclear scaffold is essential for gating rhythmic chromatin accessibility. Most importantly, loss of this architecture uncouples robustly oscillating clock protein program from its genomic and behavioral outputs. These findings reveal that circadian timekeeping is not merely a biochemical process but a spatially organized one, where nuclear topology licenses the translation of time into circadian rhythms.

cell biology↗

bioRxiv2025

Sequestration of clock proteins into repressive nuclear condensates orchestrates circadian gene repression

Circadian clocks orchestrate [~]24-hour cycles in gene expression, behavior, and physiology across most organisms1. Our recent study has revealed a striking spatial organization in the nucleus during the repression phase: core clock proteins such as Drosophila PERIOD (PER) are organized into distinct nuclear foci close to the inner nuclear envelope of clock neurons, and clock-regulated genes are similarly positioned in the nucleus2. However, the functional relevance of this subnuclear organization is unknown. Here, we investigate how the spatial organization of clock proteins and chromatin regulates circadian gene repression. Given that PER partners with TIMELESS (TIM) to enact transcriptional repression, we first investigated whether TIM is also a component of these nuclear foci. Using CRISPR-Cas9 to endogenously tag TIM with mNeonGreen, we performed high-resolution live imaging in Drosophila clock neurons. We found that TIM forms nuclear foci during the repression phase that co-localize with PER condensates, whereas TIM remains diffuse in the cytoplasm of per01 null mutants. To probe the spatial relationship between these condensates and clock-regulated genes, we combined protein imaging with fluorescence in situ hybridization (FISH) which revealed that PER/TIM condensates were positioned adjacent to, but did not overlap with, clock gene loci during the repression phase. These results suggest that core clock proteins are spatially sequestered into repressive condensates away from chromatin, providing a new framework for understanding how nuclear architecture and phase separation together orchestrate rhythmic gene expression.

cell biology↗

bioRxiv2025

An intron-based timer for circadian rhythms

Abstract: Circadian clocks regulate daily rhythms in all eukaryotes through [~]24-hour transcriptional-translational feedback loops driven by clock proteins. However, the molecular mechanisms that set the 24-hour period remain poorly defined. Here, using single-molecule imaging and nascent RNA sequencing, we uncover an unexpected RNA-based molecular timer: a single intron in the Drosophila timeless (tim) gene that regulates circadian period length by controlling mRNA localization. Strikingly, we find that [~]50% of tim mRNAs are localized to the nucleus due to inefficient post-transcriptional splicing of a single intron (which we named intron P), in contrast to other core clock transcripts that localize predominantly to the cytoplasm. CRISPR-mediated removal of intron P abolishes nuclear retention of tim transcripts, leading to accelerated TIM protein accumulation and a shortened [~]22-hour period with reduced rhythmic robustness. Remarkably, insertion of intron P alone into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, acting as a conserved checkpoint that withholds transcripts in the nucleus until splicing is complete. Finally, we identify three RNA-binding proteins, two repressors (Hrb27C and Squid) and one activator (Qkr58E-2, a Sam68 homolog), that modulate intron P splicing in a rheostat-like manner. Together, these findings establish tim intron P as the first intron-based molecular timer in circadian clocks and reveal splicing kinetics as a critical regulatory layer in temporal gene expression programs, with broad implications for other processes such as development and immunity.

molecular biology↗

bioRxiv2025

High-resolution quantification of metabolic heat output from individual live Drosophila brains

Quantitative insights into brain metabolism are essential for advancing our understanding of energy dynamics in the brain. However, current approaches for tracking brain metabolism, metabolic profiling and respirometry, provide only static snapshots of metabolite levels or lack the required resolution. Here, we develop a novel nanowatt-resolution biocalorimeter capable of real-time continuous measurements of heat output to quantitatively measure the metabolism of individual live Drosophila melanogaster brains and investigate how sex, genotype, age, and disease affect brain metabolism. We show for the first time that female brains, across multiple wild-type genotypes, exhibit a significantly higher metabolic rate ([~]10%) than male brains at a young age (<10 days old) and follow distinct metabolic trajectories across the lifespan. We also find that parkin mutants, a genetic model for Parkinsons disease, exhibit a [~]15% reduction in brain metabolic output relative to controls, revealing that defective mitophagy due to parkin deficiency affects brain metabolism. Furthermore, we measure the metabolic rate of reproductive tissues of Drosophila, highlighting the broad applicability of our biocalorimeter. Together, these advances open new avenues for investigating how tissue-specific metabolism is impacted by aging, neurodegeneration, and disease states. TeaserDirect measurement of metabolic rate of individual Drosophila brains to investigate how sex, genotype, age, and disease affect brain metabolism.

bioengineering↗

bioRxiv2023

Clock-dependent chromatin accessibility rhythms regulate circadian transcription

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. We observed significant circadian oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons. Significance StatementChromatin organization plays a critical role in gene regulation in development and in disease. In this study, we discovered robust circadian oscillations in the chromatin accessibility of regulatory elements of clock-regulated genes in Drosophila clock neurons, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. We found enrichment of E-box motifs in genes that exhibited enhanced accessibility at dusk, and enrichment of VRI/PDP1-box motifs in genes that exhibited higher accessibility at dawn. Moreover, the complete loss of chromatin accessibility rhythms in per01 mutants highlights the essential role of the Period protein in driving chromatin compaction during the repression phase. This study highlights the significance of chromatin organization in the generation of [~]24-hour circadian rhythms.

neuroscience↗

bioRxiv2022

Streamlined, cell-specific single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains

Circadian clocks are [~]24-hour timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating [~]24-hr circadian rhythms. Here we describe a streamlined single molecule Fluorescence In Situ Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of Clock, a core circadian transcription factor, in individual clock neurons in whole mount Drosophila adult brains. Specifically, we used [~]48 fluorescent oligonucleotide probes that can bind to an individual Clock mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect Clock mRNA spots in individual clock neurons. Our results demonstrate that the number of Clock mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4 hours after lights are turned on) with ~80 molecules/neuron and trough levels at ZT16 with [~]30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in Drosophila brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms.

cell biology↗

bioRxiv2022

A one-step CRISPR-based strategy for endogenous gene tagging in Drosophila melanogaster

The ability to tag endogenous genes to create fluorescent fusion proteins has revolutionized the studies of protein subcellular localization and dynamics and regulation in live cells. Here, we report a precise, rapid, and one-step endogenous gene tagging system in Drosophila melanogaster, which allows us to screen for engineered lines without the visible eye marker. Specifically, we developed an efficient screening strategy for the identification of the homologous integration events by employing a PCR-based method to characterize individual flies using only a small segment of their middle leg. Here, we detail design and construction of gRNA plasmids and donor plasmid, and methods for screening, and confirmation of engineered lines. Together, these protocols make tagging any endogenous protein in Drosophila more efficient and faster, enabling studies of a wide range of cellular processes in live cells within intact organisms.

molecular biology↗

bioRxiv2020

Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms

Circadian clocks regulate [~]24 hour oscillations in gene expression, behavior, and physiology. While the molecular and neural mechanisms of circadian rhythms are well characterized, how cellular organization of clock components controls circadian clock regulation remains poorly understood. Here, we elucidate how clock proteins regulate circadian rhythms by controlling the spatiotemporal organization of clock genes. Using high-resolution live imaging techniques we demonstrate that Drosophila clock proteins are concentrated in a few discrete foci and are organized at the nuclear envelope; these results are in contrast to longstanding expectations that clock proteins are diffusely distributed in the nucleus. We also show that clock protein foci are highly dynamic and change in number, size, and localization over the circadian cycle. Further, we demonstrate that clock genes are positioned at the nuclear periphery by the clock proteins precisely during the circadian repression phase, suggesting that subnuclear localization of clock genes plays an important role in the control of rhythmic gene expression. Finally, we show that Lamin B receptor, a nuclear envelope protein, is required for peripheral localization of clock protein foci and clock genes and for normal circadian rhythms. These results reveal that clock proteins form dynamic nuclear foci and play a hitherto unexpected role in the subnuclear reorganization of clock genes to control circadian rhythms, identifying a novel mechanism of circadian regulation. Our results further suggest a new role for clock protein foci in the clustering of clock-regulated genes during the repression phase to control gene co-regulation and circadian rhythms. SIGNIFICANCEAlmost all living organisms have evolved circadian clocks to tell time. Circadian clocks regulate [~]24-hour oscillations in gene expression, behavior and physiology. Here, we reveal the surprisingly sophisticated spatiotemporal organization of clock proteins and clock genes and its critical role in circadian clock function. We show, in contrast to current expectations, that clock proteins are concentrated in a few discrete, dynamic nuclear foci at the nuclear envelope during the repression phase. Further, we uncovered several unexpected features of clock protein foci, including their role in positioning the clock genes at the nuclear envelope precisely during the repression phase to enable circadian rhythms. These studies provide fundamental new insights into the cellular mechanisms of circadian rhythms and establish direct links between nuclear organization and circadian clocks.

neuroscience↗