Biology subjects

Asok, A.

Publications and source records attributed to Asok, A..

10 recordsLinked to original sources

bioRxiv2025

Charged Scanning Mutagenesis as a High Throughput Approach for Epitope Mapping

Identifying neutralizing epitopes is important for developing vaccines and inhibitors against viral pathogens. We describe a rapid method for epitope mapping, employing barcoded charged scanning mutagenesis libraries displayed on the yeast surface, and screening using flow cytometry coupled with deep sequencing. Prior scanning mutagenesis data suggest that mutations to a charged residue, such as Aspartic acid or Arginine, will be well tolerated at exposed positions of an antigen, and minimally affect protein stability and expression. Yet such substitutions at epitope residues strongly perturb binding to a cognate partner. We constructed an Aspartate scanning library of SARS-CoV-2 RBD and linked every mutation in the library to a defined unique barcode. The approach was used to map epitopes targeted in polyclonal sera of mice immunized with different SARS-CoV-2 immunogens. In contrast to complete mutational scans, charged scanning mutagenesis with the introduced barcoding strategy employs libraries with >50-fold lower diversity, facilitating library construction, screening, and downstream analysis, and also allowing for further multiplexing of samples, thus accelerating interaction site identification, as well as vaccine and inhibitor development.

biophysics↗

bioRxiv2025

CircRNA hypomethylation in the human amygdala implicates FKBP5 in alcohol use disorder

Genome- and phenome-wide association studies implicate the RNA demethylase FTO in alcohol use disorder (AUD), yet the RNA methylation landscape in AUD remains poorly characterized. Analyzing postmortem human basolateral amygdala (BLA) tissue, a key brain region in AUD-related behaviors, we found extensive m6A hypomethylation uniquely affecting circular RNAs (circRNAs). Notably, FKBP5-hosted circRNAs (circFKBP5s) exhibited pronounced hypomethylation correlating with elevated expression of FKBP5 mRNA isoforms. These findings were replicated in an animal model of alcohol dependence. Predictive analyses suggest that circFKBP5s influence genes involved in neurodevelopmental processes and neuronal identity. These findings uncover a novel aspect of AUD neurobiology linked to circRNA methylation.

molecular biology↗

bioRxiv2022

Circular RNAs may embed pieces of real-world sensory information into an episodic memory

For a generation, neuroscience has searched for a molecule that stores our memories across time. This search has focused on proteomic mechanisms, but less is known about RNA. Here, we identify a new persisting class of RNA associated with long-term memory - Circular RNAs. Unlike other RNAs, Circular RNAs are stable for days or longer and may provide a means for storing sensory information across time. We leveraged a differential fear conditioning paradigm whereby individual mice sample all real-world sensory inputs (i.e., auditory, visual, gustatory, olfactory, and incidental tactile) in a quasi-stochastic manner prior to receiving different intensities of an unconditioned stimulus (US) foot-shock. While Pavlovian models of learning from the 20th century were critical for understanding elemental associations, they fail to appreciate (1) what US content remains inside of a complex conditioned stimulus (CS) or response (CR - a behavioral manifestation of an episodic memory), (2) what happens when the associations involve multiple senses, and (3) what biologically happens to the real-world US. Given (1) we are constantly sampling information from our environment through all our senses and (2) the US at a given moment in time likely adds value to imprint that multisensory representation, we propose the real-world US is biologically encoded via back-spliced Circular RNAs within the cells and circuits that represent a particular episodic memory and present days later. This logic, best simplified by the equation: [Formula], allowed us to ask how the formation of similar episodic memories, which only differ in relation to the content of US information, alter Circular RNAs in the CA1 subfield of the hippocampus - a brain area critical for episodic memories. We found that stronger foot-shock USs during conditioning produce stronger memories relative to weaker USs 24-h later. Stronger memories also generalize to novel/safe environments 48-h later. Moreover, the unconditioned response is highly correlated with future CRs, suggesting (1) an understudied relationship between the strength and type of US/URs and future CRs in complex environments as well as (2) fear generalization, at least in the short-term, is associated with the embedding of additional US information. Next-generation Circular RNA sequencing 1-hr after acquisition revealed a remarkably small set of circular RNAs relative to nearly identical, yet weaker, episodic memories in CA1. Gene Ontologies for mice that formed weaker and stronger memories matched those families classically involved in weaker and stronger forms of memory across species. Preliminary in situ hybridization visually confirmed the presence of Circular RNAs in the CA1 subfield. Future experiments will examine the persistence of Circular RNAs in cells of a memory trace (i.e., engram cells; in situ hybridization) at recent (4 days) and remote (21-days) time points. Taken together with our mathematical model for multisensory learning, our data suggest that Circular RNAs do not contribute to the storage of the multisensory configural representation, but perhaps to the storage of discrete pieces of real-world sensory information related to the US that is partially embedded inside of a memory trace early-on. Importantly, in the above model for multisensory learning, the discrete USs are biologically separable from the future CS(NS+US) associations and US strength is modifiable across time. This work reveals fundamental insights into how we store pieces of real-world sensory information in an episodic memory at the biological level of the brain. One Sentence SummarycircRNAs biologically encode real-world sensory information into a long-term memory

neuroscience↗

bioRxiv2022

FAVOR: Functional Annotation of Variants Online Resource and Annotator for Variation across the Human Genome

Large-scale whole genome sequencing (WGS) studies and biobanks are rapidly generating a multitude of coding and non-coding variants. They provide an unprecedented resource for illuminating the genetic basis of human diseases. Variant functional annotations play a critical role in WGS analysis, result interpretation, and prioritization of disease- or trait-associated causal variants. Existing functional annotation databases have limited scope to perform online queries or are unable to functionally annotate the genotype data of large WGS studies and biobanks for downstream analysis. We develop the Functional Annotation of Variants Online Resources (FAVOR) to meet these pressing needs. FAVOR provides a comprehensive online multi-faceted portal with summarization and visualization of all possible 9 billion single nucleotide variants (SNVs) across the genome, and allows for rapid variant-, gene-, and region-level online queries. It integrates variant functional information from multiple sources to describe the functional characteristics of variants and facilitates prioritizing plausible causal variants influencing human phenotypes. Furthermore, a scalable annotation tool, FAVORannotator, is provided for functionally annotating and efficiently storing the genotype and variant functional annotation data of a large-scale sequencing study in an annotated GDS file format to facilitate downstream analysis. FAVOR and FAVORannotator are available at https://favor.genohub.org.

genetics↗

bioRxiv2022

Inferring bound structure and residue specific contributions to binding energetics in the Intrinsically Disordered Protein, CcdA

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration-based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a model was developed to predict mutational effects on binding affinity of IDPs that form alpha-helical structures upon binding.

biophysics↗

bioRxiv2022

Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

Social preference, the decision to interact with one member of the same species over another, is a key feature of optimizing social interactions. In rodents, social preference relies on both extrinsic factors, such as sex, strain and kinship, and intrinsic ones, such as the memory of previous encounters, which favors interactions with novel compared to familiar animals (social novelty preference). At present, it is unclear which neuronal circuits guide social preferences and whether such circuits promote social interactions with the preferred individuals or suppress interactions with the non-preferred ones. Although both the infra-limbic area of the pre-frontal cortex (ILA) and the lateral septum (LS) have been shown to support social novelty preference, the neuronal circuits and molecular mechanisms by which these brain regions interact to regulate social interactions are unknown. Here, we identify a population of inhibitory neurons in ILA that express the neuropeptide corticotropin releasing hormone (CRH) and project to the rostro-dorsal region of LS (rdLS). Release of CRH from ILA in rdLS during interactions with familiar mice disinhibits rdLS neurons, thereby suppressing interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression during the first two post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.

neuroscience↗

bioRxiv2022

Codon optimality is the primary contributor to the exceptional mutational sensitivity of CcdA antitoxin in its operonic context

Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction ([~]80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred previously.

molecular biology↗

bioRxiv2021

A multisensory circuit for gating intense aversive experiences

The ventral hippocampus (vHPC) is critical for both learned and innate fear, but how discrete projections control different types of fear is poorly understood. Here, we report a novel excitatory circuit from a subpopulation of the ventral hippocampus CA1 subfield (vCA1) to the inhibitory peri-paraventricular nucleus of the hypothalamus (pPVN) which then routes to the periaqueductal grey (PAG). We find that vCA1[->]pPVN projections modulate both learned and innate fear. Fiber photometric calcium recordings reveal that activity in vCA1[->]pPVN projections increases during the first moments of exposure to an unconditioned threat. Chemogenetic or optogenetic silencing of vCA1[->]pPVN cell bodies or vCA1[->]pPVN axon terminals in the pPVN enhances the initial magnitude of both active and passive unconditioned defensive responses, irrespective of the sensory modalities engaged by a particular innate threat. Moreover, silencing produces a dramatic impact on learned fear without affecting milder anxiety-like behaviors. We also show that vCA1[->]pPVN projections monosynaptically route to the PAG, a key brain region that orchestrates the fear response. Surprisingly, optogenetic silencing of vCA1 terminals in the pPVN titrates the level of c-Fos neural activity in the PAG differently for learned versus innate threats. Together, our results show how a novel vCA1[->]pPVN circuit modulates neuronal activity in the PAG to regulate both learned and innate fear. These findings have implications for how initial trauma processing may influence maladaptive defensive behaviors across fear and trauma-related disorders. One Sentence SummaryA multisensory gate for high intensity aversive experiences.

neuroscience↗

bioRxiv2020

The low complexity motif of cytoplasmic polyadenylation element binding protein 3 (CPEB3) is critical for the trafficking of its targets in neurons

Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs), a type of biomolecular condensate found in neurons that are necessary for local protein synthesis and are involved in long-term potentiation (LTP). Several RNA-binding proteins present in RNPs are necessary for the synaptic plasticity involved in LTP and long-term memory. Most of these proteins possess low complexity motifs, allowing for increased promiscuity. We explore the role the low complexity motif plays for RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3), a protein necessary for long-term memory persistence. We found that RNA binding and SUMOylation are necessary for CPEB3 localization to the P body, thereby having functional implications on translation. Here, we investigate the role of the low complexity motif of CPEB3 and find that it is necessary for P body localization and downstream targeting for local protein synthesis.

neuroscience↗

bioRxiv2019

Loss of function from widely distributed, synonymous mutations at single codons

Mutational tolerance inferred from laboratory-based mutational studies is typically much higher than observed natural sequence variation. Using saturation mutagenesis, we show that the ccdA antitoxin component of the ccdAB toxin-antitoxin system is unusually sensitive to mutation with over 60% of mutations leading to loss of function. Multi-base synonymous mutations at a codon display enhanced propensity to show altered phenotypes, relative to single-base ones. Such mutations modulate RNA structure, leading to altered relative translation efficiencies of the two genes in the operon, and a CcdA:CcdB protein ratio below one. These insights were used to predict and experimentally validate synonymous mutations that lead to loss of function in the unrelated relBE operon as well as the lacZ gene. Thus, synonymous mutations can have significant phenotypic effects, in the absence of overexpression or extraneous reporters. More generally, proteins are likely more sensitive to mutation than inferred from previous saturation mutagenesis studies.

molecular biology↗