Biology subjects

Schwartz, S.

Publications and source records attributed to Schwartz, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Single-Nucleotide m6A Mapping Uncovers Redundant YTHDF Function in Planarian Progenitor Fate Selection

Cell fate decisions require tight regulation of gene expression. In planarians--highly regenerative flatworms--the mRNA modification N6-methyladenosine (m6A) modulates progenitor production and fate. However, the mechanisms governing m6A deposition in the planarian transcriptome--and the role of their expanded family of YTHDF m6A reader proteins in orchestrating biological functions--remain unclear. Here, we generated the first single-nucleotide resolution map of m6A in planarians, and revealed that simple sequence rules guide m6A deposition, facilitating the flexible evolutionary gain and loss of these marks. Functional analyses of the five planarian m6A readers revealed that while individual reader expression is dispensable, together, the planarian readers regulate the production of specific progenitor lineages and overall body size. Collectively, our findings uncover a robust, redundant regulatory architecture, characterized by multiple m6A sites per gene and coordinated m6A reader expression. This architecture is essential for proper lineage resolution and provides insights into the evolutionary dynamics of the m6A landscape.

developmental biology↗

bioRxiv2024

A sequence-specific RNA acetylation catalyst

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10s catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5-CCG-3 consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10s utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

biochemistry↗

bioRxiv2024

Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2 phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

developmental biology↗

bioRxiv2024

Passive shaping of intra- and intercellular m6A dynamics via mRNA metabolism

m6A is the most widespread mRNA modification and is primarily implicated in controlling mRNA stability. Fundamental questions pertaining to m6A are the extent to which it is dynamically modulated within cells and across stimuli, and the forces underlying such modulation. Prior work has focused on investigating active mechanisms governing m6A levels, such as recruitment of m6A writers or erasers leading to either global or site-specific modulation. Here, we propose that changes in m6A levels across subcellular compartments and biological trajectories may result from passive changes in gene-level mRNA metabolism. To predict the intricate interdependencies between m6A levels, mRNA localization, and mRNA decay, we establish a differential model m6ADyn encompassing mRNA transcription, methylation, export, and m6A-dependent and independent degradation. We validate the predictions of m6ADyn in the context of intracellular m6A dynamics, where m6ADyn predicts associations between relative mRNA localization and m6A levels, which we experimentally confirm. We further explore m6ADyn predictions pertaining to changes in m6A levels upon controlled perturbations of mRNA metabolism, which we also experimentally confirm. Finally, we demonstrate the relevance of m6ADyn in the context of cellular heat stress response, where genes subjected to altered mRNA product and export also display predictable changes in m6A levels, consistent with m6ADyn predictions. Our findings establish a framework for dissecting m6A dynamics and suggest the role of passive dynamics in shaping m6A levels in mammalian systems.

systems biology↗

bioRxiv2024

Multiple ubiquitin ligases protect human genome integrity by targeting cancer-associated APOBEC3 deaminases for degradation.

HighlightsO_LIRNA-free APOBEC3 (A3) can enter the nucleus, leading to genomic mutations. C_LIO_LIThree E3 ligases specifically bind the RNA-binding domain of nuclear A3s. C_LIO_LICancer-associated A3B and A3H-I are thereby targeted for proteasomal degradation. C_LIO_LIThese E3 ligases thus act as genome guardians by limiting A3-mediated mutagenesis. C_LI APOBEC family members play crucial roles in antiviral restriction. However, certain APOBEC3 (A3) proteins drive harmful hypermutation in humans, contributing to cancer. The cancer-associated A3 proteins are capable of transiting from the cytosol to the nucleus, where they can cause genome mutations. Here, we uncover a specific set of cellular pathways that protect genomic DNA from the major cancer-associated A3 proteins. Through genetic and proteomic screening we identify UBR4, UBR5, and HUWE1 as key ubiquitin E3 ligases marking cancer-associated A3B and A3H-I for degradation, thereby limiting A3-driven hypermutation. Mechanistically, UBR5 and HUWE1 recognize unoccupied A3 RNA-binding domains, thus promoting proteasomal degradation of APOBEC3 protein that is not engaged in its antiviral cellular function. Depletion or mutation of the E3 ligases in cells and human cancer samples increases A3-driven genome mutagenesis. Our findings reveal that UBR4, UBR5, and HUWE1 are crucial factors in a ubiquitination cascade that maintains human genome stability.

cell biology↗

bioRxiv2023

VTA projections to M1 are essential for reorganization of layer 2-3 network dynamics underlying motor learning

The primary motor cortex (M1) is crucial for motor skill learning. Previous studies demonstrated that skill acquisition requires dopaminergic VTA (ventral-tegmental area) signaling in M1, however little is known regarding the effect of these inputs at the neuronal and network levels. Using dexterity task, calcium imaging, chemogenetic silencing, and geometric data analysis, we demonstrate VTA-dependent reorganization of M1 layer 2-3 during motor learning. While average activity and average functional connectivity of layer 2-3 network remain stable during learning, the activity kinetics, the correlational configuration of functional connectivity, and average connectivity strength of layer 2-3 neurons gradually transform towards an expert configuration. In addition, task success-failure outcome signaling gradually emerges. Silencing VTA dopaminergic inputs to M1 during learning, prevents all these changes. Our findings demonstrate dopaminergic VTA-dependent formation of outcome signaling and new connectivity configuration of the layer 2-3 network, supporting reorganization of the M1 network for storing new motor skills.

neuroscience↗

bioRxiv2023

txtools: an R package facilitating analysis of RNA modifications, structures, and interactions

AO_SCPLOWBSTRACTC_SCPLOWWe present txtools, an R package that enables the processing, analysis, and visualization of RNA-seq data at the nucleotide-level resolution, seamlessly integrating alignments to the genome with transcriptomic representation. txtools main inputs are BAM files and a transcriptome annotation, and the main output is a table, capturing mismatches, deletions, and the number of reads beginning and ending at each nucleotide in the transcriptomic space. txtools further facilitates downstream visualization and analyses. We showcase, using examples from the epitranscriptomic field, how a few calls to txtools functions can yield insightful and ready-to-publish results. txtools is of broad utility also in the context of structural mapping and RNA:protein interaction mapping. By providing a simple and intuitive framework, we believe that txtools will be a useful and convenient tool and pave the path for future discovery. txtools is available for installation from its GitHub repository at https://github.com/AngelCampos/txtools.

bioinformatics↗

bioRxiv2023

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2023

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and ADAR2, modulating double-stranded RNA (dsRNA) immunogenicity and recoding mRNA. The high variability in the susceptibility of different adenosines to editing begs the question of what are the determinants of substrate specificity. Here, we systematically monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into ADAR1-deleted cell lines exogenously expressing either ADAR2 or ADAR1. In both cases, structural disruptions gave rise to symmetric, strand-specific induced editing at a fixed offset, but of varying length: -26 nt for ADAR2, and -35 nt for ADAR1. We dissect the basis for the differences in offset between ADAR1 and ADAR2 via diverse mutants, domain-swaps, and ADAR evolutionary homologs, and reveal that it is encoded by the differential RNA binding domain architecture. We demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments suggestive of increased on-target and potentially decreased off-target editing. Our findings provide novel insight into the determinants guiding ADAR2 substrate selectivity and into the roles of the RNA binding domains of ADAR1 and ADAR2 in mediating differential targeting, and should facilitate the design of improved ADAR-recruiting therapeutics.

biochemistry↗

bioRxiv2022

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the {beta}-glucan receptor Dectin-1, and respond to commensal fungi and {beta}-glucans. In phagocytes, Dectin-1 mediates LC3 associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of {beta}-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose {beta}-glucan-containing fungal particles via LAP. Zymosan ({beta}-glucan particle) and Heat-killed and UV inactivated C. albicans were phagocytosed by monolayers of human colonic (n=18) and ileal (n=4) organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases. Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.

immunology↗

bioRxiv2022

Tm-22 resistance targets a conserved cysteine essential for tobacco mosaic virus (TMV) movement

The tomato Tm-22 gene was considered one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV). However, an emerging tobamovirus, Tomato brown rugose fruit virus (ToBRFV), has overcome Tm-22, damaging tomato production worldwide. Tm-22 encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MPToBRFV) enabled the virus to overcome Tm-22- mediated resistance. Yet, it was unknown how Tm-22 remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that the presence of a conserved cysteine (C68) in the MP of TMV (MPTMV) is both sufficient to trigger Tm-22 resistance and essential for viral movement. Substitution of MPToBRFV amino acid H67 with the corresponding amino acid in MPTMV (C68) activated Tm-22-medited resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV, and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell, and subcellular movement analysis showed that C68 is required for the movement of TMV, by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-22 immune activation could explain how TMV was unable to overcome this resistance for such a long period.

plant biology↗

bioRxiv2022

Scream's roughness confers a privileged access to the brain during sleep

During sleep, recognizing threatening signals is crucial to know when to wake up and when to continue vital sleep functions. Screaming is perhaps the most salient and efficient signal for communicating danger at a distance or in conditions of limited visibility. Beyond the intensity or the pitch of the sound, rapid modulations of sound pressure in the so-called roughness range (i.e. 30-150 Hz) are particularly powerful in capturing attention and accelerating reactions. Roughness is an acoustic feature that characterizes alarm signals such as screams. However, whether rough sounds are also processed in a privileged manner during sleep is unknown. We tested this hypothesis by stimulating sleeping human participants with low-intensity screams and neutral calls. We found that screams trigger more reliable and better time-locked responses in wakefulness and NREM sleep. In addition, screams boosted sleep spindles, suggesting elevated stimulus salience. The increase in sleep spindle power was linearly proportional to the roughness of vocalizations, but not to their pitch. These findings demonstrate that, even at low sound intensity, screams roughness conveys stimulus relevance and enhances processing in both the waking and sleeping states. Preserved differential neural responses based on stimulus salience may ensure adaptive reactions -and ultimately survival- in a state where the brain is mostly disconnected from external inputs.

neuroscience↗

bioRxiv2022

Exon-intron architecture determines mRNA stability by dictating m6A deposition

N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we uncover that m6A deposition is not selective. Instead, m6A distribution is exclusion-based: m6A-consensus harboring sites are methylated by default, unless they are within a window of up to [~]200 nt from an exon-intron junction. A simple model, relying exclusively on presence of m6A motifs and exon-intron architecture allows high accuracy recapitulation of experimentally-measured m6A profiles and of all m6A hallmarks. We further establish that m6A serves as the long-sought mechanism underlying the strong association between exon-intron architecture and mRNA stability. Our findings establish a mechanism by which the memory of nuclear RNA splicing is covalently etched on an mRNA, in the form of m6A, and determines its cytoplasmic stability, with broad implications on the regulation, function, and evolution of both m6A and mRNA stability.

molecular biology↗

bioRxiv2022

Reinstatement of emotional associations during human sleep: an intracranial EEG study

The scientific literature suggests that emotional memories benefit from a privileged consolidation over neutral memories. This effect extends to consolidation processes that occur during sleep. Indeed, during sleep, a complex set of oscillations (namely slow-oscillations, theta rhythm and spindles) mediates the communication between brain regions involved in the long-term integration of memories. However, whether sleep oscillations may contribute to the reactivation and consolidation of emotional memories in humans is still unclear. Because non-invasive electroencephalography (EEG) has limited access to deep brain regions implicated in memory and emotion (e.g., hippocampus, amygdala, orbitofrontal cortex), here we recorded EEG signal from these brain regions using intracranial electrodes placed in medically-resistant epileptic patients in the context of presurgical investigation. During wakefulness, we presented the patients with emotional (i.e., humorous) vs emotionally neutral pictures paired with a sound. Then, we tested for the reinstatement of emotional-associations by delivering the sound during a subsequent period of sleep. We found that the reactivation of emotional (compared to neutral) memories during sleep enhanced slow-oscillation and spindle activity in the orbitofrontal cortex, paralleled with an increase in theta connectivity between the hippocampus and the orbitofrontal cortex. In addition, we observed that the theta response to emotional memories reactivated at subsequent wake was different than for neutral memories, suggesting a change in memory traces with targeted memory reactivation. These data suggest that consolidation of emotional events during sleep is due to a larger expression of sleep features (in the slow-oscillation, theta and sigma frequency bands) and that the mechanisms of brain plasticity also take place in emotional brain regions during NREM sleep.

neuroscience↗

bioRxiv2022

Cognitive exertion affects the appraisal of one's own and other people's pain

Correctly evaluating others pain is a crucial prosocial ability, especially relevant for the healthcare system. In clinical settings, caregivers assess their patients pain under high workload and fatigue, often while dealing with competing information/tasks. However, the effect played by such cognitive strain in the appraisal of others pain remains unclear. Following embodied accounts that posit a shared representational code between self and others states, it could be hypothesized that the representation of peoples pain might be influenced by cognitive exertion similarly to first-hand experiences. Fifty participants underwent one of two demanding tasks, involving either working memory (Experiment 1: N-Back task) or cognitive interference (Experiment 2: Stroop task). After each task, participants were exposed to painful laser stimulations at three intensity levels (low, medium, high), or video-clips of patients experiencing three intensity levels of pain (low, medium, high). Participants rated the intensity of each pain event on a visual analogue scale. We found that the two tasks influenced rating of both ones own and others pain, by decreasing the sensitivity to medium and high events. This was observed either when comparing the demanding condition to a control (Stroop), or when modelling linearly the difficulty/performance of each depleting task (N-Back). We provide converging evidence that cognitive exertion affects the subsequent appraisal of ones own and likewise others pain. Healthcare personnel should be aware that high workload might alter their cognitive abilities. Perspectivethis research shows that cognitive effort aftereffects impact negatively the assessment of of medium/high pain in others, reminiscently to what was observed in first-hand experiences. Healthcare professionals should be aware that high workload and severe cognitive fatigue could affect their diagnostic skills.

neuroscience↗

bioRxiv2022

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. Establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. Here we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically-expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g. pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2022

Evaluation of Cognitive Function in the Dog Aging Project: Associations with Baseline Canine Characteristics

Canine Cognitive Dysfunction (CCD) is a neurodegenerative disease in aging dogs. It has been described previously in relatively small cohorts of dogs using multiple different rating scales. This study aimed to use a minimally modified CCD rating scale developed by previous researchers to describe the prevalence of CCD more thoroughly in a large, nationwide cohort of companion dogs participating in the Dog Aging Project (DAP). Associations between various canine characteristics, predicted lifespan quartiles, and CCD were examined using univariable and multivariable logistic regression models and Receiver Operating Curve (ROC) analysis. When controlling for all other characteristics, the odds of CCD increased 52% with each additional year of age. Among dogs of the same age, health status, breed type, and sterilization status, odds of CCD were 6.47 times higher in dogs who were not active compared to those who were very active. When controlling for age, breed type, activity level, and other comorbidities, dogs with a history of neurological, eye, or ear disorders had higher odds of CCD. Lifespan quartile analysis showed excellent discriminating ability between CCD positive and negative dogs. Weight-based lifespan quartile estimation could therefore serve as a tool to inform CCD screening by veterinarians.

animal behavior and cognition↗

bioRxiv2022

Cardio-audio synchronization elicits prediction in auditory sequences during human wakefulness and sleep

The human brain can infer temporal regularities in auditory sequences with fixed sound-to-sound intervals and in pseudo-regular sequences where sound onsets are locked to cardiac inputs. Here, we investigated auditory and cardio-audio regularity encoding during sleep, when reduced vigilance may result in altered bodily and environmental stimulus processing. Using electroencephalography and electrocardiography in healthy volunteers (N=26) during wakefulness and sleep, we measured the response to unexpected sound omissions within three auditory regularity conditions: synchronous, where sound and heartbeat are temporally locked, isochronous, with fixed sound-to-sound intervals, and a control condition without specific regularity. During wakefulness and all sleep stages, the cardiac signal following sound omissions exhibited a deceleration over time in the synchronous condition only. At the neural level, both the synchronous and isochronous sequences gave rise to a neural omission response in wakefulness and N2 sleep. Regularity encoding in the synchronous and isochronous conditions was characterized by a modulation of the background oscillatory activity in N2 sleep, outlining a possible mechanism through which the brain aligns periods of high neuronal excitability to the expected sound onset. The violation of auditory and cardio-audio regularity elicits cardiac and neural surprise responses across vigilance stages. Significance StatementAcross vigilance states, the human brain can generate predictions about the future based on past sensory regularities. While this is evident for environmental stimuli, the role of bodily signals in forming sensory prediction remains unknown. Here, we show that the human brain utilizes the temporal relationship between cardiac and auditory inputs in order to anticipate upcoming sounds during wakefulness and sleep. After presenting sounds in synchrony with the ongoing heartbeat, a sound omission elicited both a heartbeat deceleration and a prediction error signal as measured by the electroencephalographic response. Heartbeat signals support auditory regularity encoding during sleep and wakefulness, highlighting one mechanism for optimizing the detection of unexpected stimuli by taking advantage of the continuously monitored cardiac signals.

neuroscience↗