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Kadener, S.

Publications and source records attributed to Kadener, S..

18 recordsLinked to original sources

"Remote and partial clocks expand the circadian neuronal network, driving widespread molecular rhythmicity in Drosophila"

Circadian clocks orchestrate daily physiology and behavioral rhythms, yet the extent to which cells lacking canonical clock components exhibit robust temporal regulation remains unclear. Here we addressed this question using a two-step strategy. First, we systematically mapped core clock gene expression at single-cell resolution across the Drosophila brain and body. Second, leveraging these data and performing targeted experiments, we uncovered cells that lack most or all canonical clock components yet display strong mRNA rhythms. We found that lamina wide-field neurons show high-amplitude tim mRNA cycling even in constant darkness conditions (DD) despite minimal expression of other clock genes, suggesting a partial or noncanonical oscillator. In addition, C2 and C3 optic lobe neurons, which do not express core clock components, exhibit hundreds of circadian and daily cycling transcripts. Notably, circadian rhythms in C3 neurons coincide with oscillations of activity-regulated genes (ARGs), whereas C2 neurons cycle independently of ARGs, indicating distinct non-cell-autonomous mechanisms. These findings reveal a spectrum of circadian regulation, from autonomous to remote, input-driven rhythms and expand the circadian landscape to include strategies for generating and distributing temporal information across neuronal and non-neuronal cell types.

neuroscience↗

Neuronal development shapes activity-dependent gene expression in a stimulus-specific manner

Neuronal activity-dependent gene expression is fundamental to a wide variety of brain functions. However, how development progress and stimulation modalities specifically affect neuron transcription is not fully understood. In this work, we first investigate the influence of development on neuronal firing and activity-driven transcription. We used an RNA sequencing approach over 7 days in vitro (DIV) or mature 21 DIV neurons, comparing neuronal depolarization with potassium chloride (KCl) versus Biccuculine application, a synaptic modality to induce neuronal activity. To further investigate how different activity patterns influence gene transcription in mature neurons, we compared global gene expression in neurons treated with three different and extensively used activation protocols: KCl, Bicuculine (Bic), and TTX withdrawal (TTXw). Our results demonstrate a strong influence of development on activity-dependent gene expression, and showed that different patterns of neuronal activity induce different transcriptional profiles and exhibit distinct temporal dynamics for the same genes. These findings offer novel insights into the complex relationship between neuronal activity and gene expression, shedding light on the context-dependent nature of activity-dependent transcriptional responses.

neuroscience↗

Tissue-Like Multicellular Development Triggered by Mechanical Compression in Archaea

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once observed in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity become akin to animal tissues and give rise to two cell types--radial peribasal and central apicobasal cells-- with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

cell biology↗

Circular RNAs exhibit exceptional stability in the aging brain and serve as reliable age and experience indicators

Circular RNAs (circRNAs) comprise a large class of stable RNAs produced through backsplicing. While circRNAs have been shown to be very stable in cell culture, it is unknown how stable they are in vivo. Interestingly, studies across various animal systems demonstrated that circRNAs levels increase with age in neural tissue. However, the underlying reasons for this age-related accumulation are still unclear. To address these questions, we profiled circRNAs from fly heads at six timepoints across their lifespan. We found that circRNA levels increase linearly with age, independent of changes in mRNA levels, overall transcription, intron retention, or host genes splicing. This indicates that the age-related accumulation is attributed to their extraordinary stability in neural tissue rather than changes in biosynthesis. Furthermore, exposure to environmental stimuli like different temperatures, resulted in the increase but not decrease of circRNAs subsets, further confirming their stability in vivo. This exceptional stability implies that circRNAs can serve as markers of environmental experience. Indeed, flies subjected to a ten-day regimen at 29{degrees}C exhibit higher levels of specific brain circRNAs even six weeks after returning to standard conditions, indicating that circRNAs can reveal past environmental stimuli. Additionally, circRNA half-life measurements revealed values exceeding 20 days, with some showing no degradation over the animals lifetime. These findings demonstrate the extreme stability of circRNAs in vivo and their use as markers for aging, stress and life experiences.

neuroscience↗

Organismal landscape of clock cells and circadian gene expression in Drosophila

BackgroundCircadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in Drosophila some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF). Despite these findings and the ample knowledge of the molecular clockwork, it is still unknown how circadian gene expression in Drosophila is achieved across the body. ResultsHere, we used single-cell and bulk RNAseq data to identify cells within the fly that express core-clock components. Surprisingly, we found that less than a third of the cell types in the fly express core-clock genes. Moreover, we identified Lamina wild field (Lawf) and Ponx-neuro positive (Poxn) neurons as putative new circadian neurons. In addition, we found several cell types that do not express core clock components but are highly enriched for cyclically expressed mRNAs. Strikingly, these cell types express the PDF receptor (Pdfr), suggesting that PDF drives rhythmic gene expression in many cell types in flies. Other cell types express both core circadian clock components and Pdfr, suggesting that in these cells, PDF regulates the phase of rhythmic gene expression. ConclusionsTogether, our data suggest three different mechanisms generate cyclic daily gene expression in cells and tissues: canonical endogenous canonical molecular clock, PDF signaling-driven expression, or a combination of both.

molecular biology↗

Artificially stimulating retrotransposon activity increases mortality and accelerates a subset of aging phenotypes in Drosophila

Transposable elements (TE) are mobile sequences of DNA that can become transcriptionally active as an animal ages. Whether TE activity is simply a byproduct of heterochromatin breakdown or can contribute towards the aging process is not known. Here we place the TE gypsy under the control of the UAS GAL4 system to model TE activation during aging. We find that increased TE activity shortens the lifespan of male D. melanogaster. The effect is only apparent in middle aged animals. The increase in mortality is not seen in young animals. An intact reverse transcriptase is necessary for the decrease in lifespan implicating a DNA mediated process in the effect. The decline in lifespan in the active gypsy flies is accompanied by the acceleration of a subset of aging phenotypes. TE activity increases sensitivity to oxidative stress and promotes a decline in circadian rhythmicity. The overexpression of the Forkhead-box O family (FOXO) stress response transcription factor can partially rescue the detrimental effects of increased TE activity on lifespan. Our results provide evidence that active TEs can behave as effectors in the aging process and suggest a potential novel role for dFOXO in its promotion of longevity in D. melanogaster.

molecular biology↗

An integrative omics approach reveals posttranscriptional mechanisms underlying circadian temperature compensation

A defining property of circadian clocks is temperature compensation, characterized by the resilience of circadian free-running periods against changes in environmental temperature. As an underlying mechanism, the balance or critical reaction hypothesis have been proposed. While the former supposes a temperature-dependent balancing of reactions with opposite effects on circadian period, the latter assumes an insensitivity of certain critical period determining regulations upon temperature changes. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3-end cleavage and polyadenylation, abolishes circadian temperature compensation in U-2 OS cells. We apply a combination of 3-End-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3 UTR length as well as gene and protein expression between wild type and CPSF6 knock-down cells and their dependency on temperature. Analyzing differential responses upon temperature changes in wild type and CPSF6 knockdown cells reveals candidate genes underlying circadian temperature compensation. We identify that eukaryotic translation initiation factor 2 subunit 1 (EIF2S1) is among these candidates. EIF2S1 is known as a master regulator of cellular stress responses that additionally regulates circadian rhythms. We show that knockdown of EIF2S1 furthermore impairs temperature compensation, suggesting that the role of CPSF6 in temperature compensation may be mediated by its regulation of EIF2S1.

genomics↗

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1,000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

molecular biology↗

The chromatin factor ROW cooperates with BEAF-32 in regulating long-range genes

Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in regulating chromatin loops. Yet, how chromatin loops contribute to transcription regulation is still not clear. Here we show that Relative-of-WOC (ROW) is essential for the long-range transcription regulation mediated by the Boundary Element-Associated Factor of 32kD (BEAF-32). We found that ROW physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein BEAF-32. The co-localization happens at TAD boundaries where ROW, through its AT-hooks motifs, binds AT-rich sequences flanked by BEAF-32 binding sites and motifs. Knockdown of row resulted in downregulation of genes that are long-range targets of BEAF-32 and bound indirectly by ROW (without binding motif). Analysis of high- throughput chromosome conformation capture (Hi-C) data revealed long-range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, our results show cooperation between BEAF-32 and the ROW complex, which includes HP1 proteins, to regulate the transcription of developmental and inducible genes by chromatin loops.

genomics↗

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

Neurons draw on alternative splicing for their increased transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. We found that in Drosophila, this splicing program is restricted to neurons by the post-transcriptional processing of the enhancer of microexons (eMIC) domain in Srrm234 by Elav and Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell-type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. Remarkably, we found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient post-transcriptional programs can evolve independently in different phyla to impact distinct cellular modules while maintaining cell-type specificity.

molecular biology↗

CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells.

Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.

molecular biology↗

An in vivo strategy for knockdown of circular RNAs

Exonic circular RNAs (circRNAs) are highly abundant and evolutionarily conserved RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize miRNA-derived shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in the fly nervous system. There were no effects on levels of the linear RNA or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, we found that downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cell levels.

molecular biology↗

Host-derived Circular RNAs Display Proviral Activities in Hepatitis C Virus - Infected Cells

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Surprisingly, circPSD3 also inhibited the cellular nonsense-mediated decay (NMD) pathway in liver cells. Thus, enhanced abundance of circPSD3 in virus-infected cells aids in viral replication and likely contributes to the known inhibition of NMD in HCV-infected cells. Findings from the global analyses of the circular RNA landscape argue pro-, and likely, anti-viral functions are executed by circRNAs that modulate both viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis. Author SummaryUsually, cells are infected by one or a few virus particles that carry genomes with limited expression capacity. Thus, the expression of viral genomes has to compete with a sea of cellular components that aid in viral translation, replication and virion production. Depending on their lifestyle, viruses have evolved to avoid or to subvert cellular pathways, especially those that display anti-viral functions. Host-derived circular RNA molecules have recently been discovered in the cytoplasm of cells, although, as-of yet, few functions have been assigned to them. Here, we describe alterations in the circular RNA landscape in hepatitis C virus-infected liver cells. Up-regulated and down-regulated circular RNAs were identified, and three of the upregulated RNAs were shown to promote HCV infection. One of them, circPSD3, inhibited the cellular nonsense-mediated RNA decay that is a powerful antiviral response in infected cells. Because circular RNAs are more stable than linear RNAs, they may have important functions during viral infection, dictating the outcomes of innate immune responses and viral pathogenesis.

microbiology↗

A comprehensive pipeline for accurate annotation and quantification of circRNAs

Identification and quantification of circular RNAs (circRNAs) depends strongly on the utilized computational pipeline. Here we describe an integrative approach for accurate annotation and quantification of circRNAs. First, we utilize several circRNA-identification pipelines to annotate circRNAs in a given organism. Second, we build a short sequence index that is used to search the unaligned RNA-seq reads. Our approach allows full annotation of circRNAs with fewer false positives and negatives than any individual pipeline or combination of them. Moreover, our approach is more sensitive than any individual pipeline and allows more accurate quantification and larger number of differentially expressed circRNAs.

bioinformatics↗

Transcription of a plasticity gene is activated by neuronal hyperpolarization

Long term synaptic plasticity requires transcriptional changes in response to neuronal activity. While neuronal activity rapidly increases transcription of activity-regulated genes, it is less clear how neurons respond to neuronal inactivity. The plasticity gene Pura encodes a Rho1 GEF whose rhythmic expression drives 24 hour rhythms in the retraction of the projections of Drosophila LNv circadian pacemaker neurons. We found that Pura transcription in LNvs is repressed by neuronal activity and induced by neuronal inactivity - the opposite of canonical activity-regulated genes. We identified the Pax6 transcription factor Toy as the relevant activator of Pura transcription, and found that toy transcription itself is induced by neuronal inactivity and by reducing intracellular calcium. Thus we propose that neurons contain complementary gene expression programs that are activated by either increased or decreased neuronal activity.

neuroscience↗

Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila

Circadian rhythms are generated by the cyclic transcription, translation and degradation of clock genes, including timeless (tim). Currently, little is known about the mechanisms by which the circadian clock senses and adapts to temperature changes. Here we show that temperature dramatically changes the splicing pattern of tim. We found that at 18{degrees}C TIM protein levels are diminished due to the induction of two cold-specific splicing isoforms (tim-cold and tim-short&cold). At 29{degrees}C, another isoform, tim-Medium is strongly upregulated. We found that this isoform switching mechanism allows flies to regulate the levels and activity of TIM by setting miRNA-dependent thresholds for expression as well as by expressing isoforms with specific functions. Flies in which the production of tim-short&cold is abrogated display altered patterns of locomotor activity and altered tim expression. Interestingly, the introns of tim carry the information for the temperature sensitivity, suggesting that tim splicing per se is the temperature sensor.

neuroscience↗

PSI controls tim splicing and circadian period in Drosophila

The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to both post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. To have a better understanding of the role and mechanisms of circadian post-transcriptional regulation, we targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 genes we identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI downregulation shortens the period of circadian rhythms both in the brain and in peripheral tissues. Interestingly, we found that PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Moreover, the period of circadian behavior was insensitive to PSI downregulation when flies could produce functional TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, we conclude that PSI regulates the period of Drosophila circadian rhythms through its modulation of the tim splicing pattern.

neuroscience↗

An in vivo knockdown strategy reveals multiple functions for circMbl

Circular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl. Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo.\n\nSIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

molecular biology↗