Biology subjects

Rajshekar, S.

Publications and source records attributed to Rajshekar, S..

5 recordsLinked to original sources

bioRxiv2026

ORC binding to Heterochromatin Protein 1 through intrinsically disordered regions is required for heterochromatin structure and function

The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster. Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC-HP1 interactions, which also requires HP1 dimerization. Disrupting ORC-HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.

molecular biology↗

bioRxiv2025

Alternative Splicing in TRPA1 Drives Sensory Adaptation to Electrophiles in Drosophilids

Behaviors are among the first traits to evolve as animals enter new niches, but their molecular bases are poorly understood. To address this gap, we used the mustard-feeding drosophilid fly Scaptomyza flava, which feeds on toxic Brassicales plants like wasabi that produce noxious, electrophilic isothiocyanates (ITCs or mustard oils). We found that S. flava exhibits dramatically reduced behavioral sensitivity to allyl isothiocyanate (AITC) compared to its microbe-feeding relatives Scaptomyza pallida and Drosophila melanogaster. We hypothesized that molecular evolution of the "wasabi receptor" TRPA1, known to detect ITCs in flies, could explain this loss of aversion. Our experiments revealed three interconnected evolutionary genetic changes consistent with this hypothesis. First, TRPA1 was expressed in labellar tissues of S. flava at the lowest levels among the three species, at a nearly four-fold lower level than in D. melanogaster. Second, S. flava expressed a higher proportion of TRPA1 splice variants previously reported to be less sensitive to chemical stimulus. Third, we identified amino acid substitutions in S. flava that could influence the structure of intracellular domains of TRPA1. To test the functional salience of these mechanisms in vitro and in vivo, we validated TRPA1 splicing isoforms using Xenopus oocyte electrophysiology and the GAL4/UAS system in D. melanogaster. Single TRPA1 isoform electrophysiology in vitro revealed evolution of the channel in the S. flava lineage towards reduced electrophile sensitivity. Ectopic expression of S. flava TRPA1 in D. melanogaster also consistently conferred weaker AITC sensitivity in vivo than expression of its orthologues, although this did not fully recapitulate differences in wild-type phenotypes between species, suggesting other molecular mechanisms were involved. To address this, we explored the consequences of isoform co-expression using oocyte electrophysiology. We found that enrichment of electrophile-insensitive TRPA1 splicing isoforms as observed in the salient S. flava sensory organs additively reduced cellular responses to AITC, which could further contribute to reduced electrophile aversion. Our findings illuminate how expression differences, protein structural changes, and especially alternative splicing, together can drive sensory evolution as animals behaviorally adapt to toxic new niches.

evolutionary biology↗

bioRxiv2024

Odorant receptors tuned to isothiocyanates in Drosophila melanogaster and their evolutionary expansion in herbivorous relatives

Plants release defense volatile compounds that can deter herbivores. Among them are electrophilic toxins, such as isothiocyanates from mustard plants, that activate pain receptors by contact (i.e. taste) in many animals, including Drosophila melanogaster. While specialist insects have evolved strategies to tolerate toxicity and use mustard plants as hosts, it is unclear whether non-specialist insects detect and avoid electrophilic toxins via olfaction. To address this, and to understand if specialized insects co-opted these toxic compounds as hostplant olfactory cues, we leveraged closely related drosophilid species, including the microbe-feeding D. melanogaster and Scaptomyza pallida, and the mustard-feeding specialist S. flava. In olfactory assays, D. melanogaster exposed to allyl isothiocyanate volatiles were rapidly immobilized, demonstrating the high toxicity of this wasabi-derived compound to non-specialists. Through single sensillum electrophysiological recordings from olfactory organs and behavioral assays, we identified an Olfactory receptor (Or) necessary for volatile detection and behavioral aversion to allyl isothiocyanate in D. melanogaster. RNA sequencing and heterologous expression revealed that S. flava possess lineage-specific, triplicated homologs of this Or, and that each paralog exhibited broadened and distinct sensitivity to isothiocyanate compounds. Using AlphaFold2 modeling, site-directed mutagenesis and electrophysiological recordings, we identified two critical amino acid substitutions that changed the sensitivity of these paralogs from fruit-derived odors to isothiocyanates in the mustard specialist S. flava. Our findings show that non-specialists can detect electrophiles via olfaction, and that their olfactory systems can rapidly adapt to toxic hostplant niches through co-option and duplication of ancestral chemosensory genes with few amino acid changes.

evolutionary biology↗

bioRxiv2023

Affinity hierarchies underlie the co-assembly of nucleolar and heterochromatin condensates in Drosophila embryos

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. Nucleoli are the sites of ribosome synthesis, while the repeat-rich PCH is essential for chromosome segregation, genome stability, and transcriptional silencing. How and why these two distinct condensates co-assemble is unclear. Here, using high-resolution live imaging of Drosophila embryogenesis, we find that de novo establishment of PCH around the nucleolus is highly dynamic, transitioning from the nuclear edge to surrounding the nucleolus. Eliminating the nucleolus by removing the ribosomal RNA genes (rDNA) resulted in increased PCH compaction and subsequent reorganization into a toroidal structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or neocondensates, including enrichment in the PCH toroidal hole. Combining these observations with physical modeling revealed that nucleolar-PCH associations can be mediated by a hierarchy of interaction strengths between PCH, nucleoli, and amphiphilic protein(s) that have affinities for both nucleolar and PCH components. We validated this model by identifying a candidate amphiphile, a DEAD-Box RNA Helicase called Pitchoune, whose depletion or mutation of its PCH interaction motif disrupted PCH-nucleolar associations. Together, this study unveils a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrates that nucleoli are required for normal PCH organization, and identifies Pitchoune as an amphiphilic molecular link required for PCH-nucleolar associations.

cell biology↗

bioRxiv2018

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome

Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity.

molecular biology↗