Biology subjects

Sahadevan, S.

Publications and source records attributed to Sahadevan, S..

9 recordsLinked to original sources

bioRxiv2026

Identification of RNA Targets of Classical and Non-Canonical RNA-binding Proteins by soniCLIP

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/745202v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@52e3edorg.highwire.dtl.DTLVardef@1f21637org.highwire.dtl.DTLVardef@909ee8org.highwire.dtl.DTLVardef@b0c907_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG Crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) is widely used to identify the RNA targets of RNA-binding proteins (RBPs). However, its application to non-canonical RBPs lacking canonical RNA-binding domains and frequently displaying low or transient RNA occupancy, is limited by low signal-to-noise ratios, high input requirements and error-prone ligation steps during library preparation. To overcome these limitations, we developed soniCLIP, a streamlined CLIP-seq workflow that replaces RNase-mediated RNA fragmentation with sonication and uses a ligation-free strategy for library construction. soniCLIP is optimized for reproducible identification of enriched RBP-associated RNA regions from limited starting material. We benchmarked soniCLIP against the widespread eCLIP approach and observed reproducible recovery of known RBP-associated regions and target recovery comparable to ENCODE eCLIP, while requiring only 10% (500 {micro}g) of protein input. We further applied soniCLIP to the glycolytic enzyme and non-canonical RBP pyruvate kinase M2 (PKM2). We identified 197 significantly enriched RNA regions and validated selected targets by RIP-qRT-PCR and in vitro binding assays. By combining reduced input requirements, high reproducibility, a shortened 3.5-day workflow and the elimination of gel-based purification, soniCLIP provides an efficient and robust approach for the identification of RNA targets of canonical and non-canonical RBPs.

molecular biology↗

bioRxiv2026

Increased expression of Cd74 and MHC II genes by aortic macrophages links atherosclerosis with aging in mice

Atherosclerosis is a chronic inflammatory condition of the arteries leading to myocardial infarction, ischemic stroke and peripheral arterial diseases. Atherosclerotic plaques, which obstruct blood flow contain functionally diverse macrophage populations including pro-atherogenic and atheroprotective subsets. Aging is a major risk factor of atherosclerosis, but the mechanism underlying aging associated risk of atherosclerosis is unclear. Here, using integrated single-cell RNA sequencing data analysis, we demonstrate that specific monocyte and macrophage subsets are enriched in atherosclerotic plaques and the aging aorta of mice. We also show that Cd74 and MHCII genes such as H2-Aa, H2-Ab1, H2-Eb1, and H2-DMb1 are consistently upregulated in the monocytes and macrophages from atherosclerotic plaques, the aging aorta, and the aging bone marrow of mice. Our experimental data also show increased expression of CD74 surface protein by aortic macrophages and bone marrow monocytes from aged mice. In addition, we show increased CD74 expression by RAW264.7 mouse macrophages and THP1 human monocytes following ox-LDL stimulation in vitro. Finally, we demonstrate that monocytes from aged mice adhere more to aortic endothelial cells in co-cultures. Thus, increased monocyte adhesion to endothelial cells may explain the enhanced proportion of specific monocyte and macrophage subsets in the aging aorta, inducing a pre-atherosclerotic condition.

immunology↗

bioRxiv2026

Muscle LIM Protein is a Non-Canonical RNA-Binding Protein that Engages in RNA-Dependent Cytoskeletal Assemblies

Muscle LIM Protein (MLP) is a key component of the cardiomyocyte Z disc that is essential for sarcomere integrity and cardiac homeostasis. Accordingly, loss of MLP function results in (cardio)myopathy phenotypes in animal models and human patients. In this study, we present the first RNA-binding proteome (RBPome) of human cardiomyocytes using enhanced RNA interactome capture (eRIC). Data integration with existing RBPome datasets identified MLP as an evolutionary conserved and previously unrecognized non-canonical RNA-binding protein (RBP) in cardiomyocytes. Using complementary biochemical approaches, we confirmed direct RNA association of MLP. Moreover, RNA integrity was required for MLP interactions with cytoskeletal proteins and for the formation of higher-order MLP-containing assemblies, revealed by co-immunoprecipitation and RNA-dependent sedimentation profiling. Subcellular fractionation and imaging analyses indicated that MLPs association with cytoskeletal complexes may be weaker and more dynamic compared to core sarcomere proteins like -actinin. Domain mapping identified two glycine-rich regions within MLP as RNA contact sites, and RNA-binding-deficient MLP mutants showed impaired association with cytoskeletal complexes. Together, our findings suggest RNA binding as a regulated property of MLP and uncover an RNA-dependent layer of cytoskeletal organization in cardiomyocytes.

molecular biology↗

bioRxiv2025

Refate identifies chemical compounds to target trans-regulatory networks for cellular conversion

Identifying chemical compounds that target trans-regulatory networks (TRNs) underlying molecular programs of cells for directed cellular conversion (i.e. differentiation, reprogramming, transdifferentiation, and dedifferentiation) is a key step towards advancing regenerative medicine. Recent innovations in single-cell omics technologies enabled high-resolution profiling of TRNs that govern cell identity and cell-fate decisions. Here, we introduce Refate, a computational framework that integrates large-scale multimodal single-cell atlas data to quantify cell propensity of genes, together with six drug databases, to identify chemical compounds that target TRNs for directed cellular conversion. The reconstructed TRNs, including protein-protein interactions and gene regulatory networks, alongside chemical compounds that drive the cellular conversion provide greater biological interpretability and improve efficiency and efficacy. We evaluated Refate by testing its ability to uncover known transcription factors and chemical compounds validated in experimental conversions of various cell types. Furthermore, we experimentally validated the attribute of several novel chemical compounds identified by Refate for enhancing the conversion of human embryonic stem cells to human cranial neural crest cells. Together, these findings demonstrate Refate as an effective tool for discovering chemical compounds that target TRNs to enable cellular conversion, advancing efforts towards regenerative medicine. HighlightsO_LIRefate quantifies genes for cellular conversion using multimodal single-cell atlases C_LIO_LIRefate uncovers trans-regulatory networks (TRNs) underlying cellular conversion C_LIO_LIRefate identifies chemical compounds that target TRNs for cellular conversion C_LIO_LIValidation of Refate identified chemical compounds for hESC to hCNCC conversion C_LI

developmental biology↗

bioRxiv2022

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a powerful method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can profoundly deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false-positive rates. Here, we present the R/Bioconductor package DEWSeq that makes full use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing (2.3-fold median). The improvement not only relates to the number of binding sites (e.g., 3.1-fold of known motifs for RBFOX2), but also their subcellular localisation (e.g., 1.9-fold of mitochondrial genes for FASTKD2) and structural targets (e.g., 2.2-fold increase of stem-loop regions for SLBP). DEWSeq therefore shows promise as an improved processing method for eCLIP protein-RNA interaction data.

molecular biology↗

bioRxiv2022

Small non-coding RNA Interactome Capture reveals pervasive, carbon source-dependent tRNA engagement of yeast glycolytic enzymes

Small non-coding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small non-coding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small non-coding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nucleotides in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognised interactions.

molecular biology↗

bioRxiv2022

TDP-43 oligomerization and RNA binding are codependent but their loss elicits distinct pathologies

Aggregation of the RNA-binding protein TDP-43 is the main common neuropathological feature of TDP-43 proteinopathies. In physiological conditions, TDP-43 is predominantly nuclear and contained in biomolecular condensates formed via liquid-liquid phase separation (LLPS). However, in disease, TDP-43 is depleted from these compartments and forms cytoplasmic or, sometimes, intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Here, we show that self-oligomerization and RNA binding cooperatively govern TDP-43 stability, functionality, LLPS and cellular localization. Importantly, our data reveal that TDP-43 oligomerization is connected to, and conformationally modulated by, RNA binding. Mimicking the impaired proteasomal activity observed in patients, we found that TDP-43 forms nuclear aggregates via LLPS and cytoplasmic aggregates via aggresome formation. The favored aggregation pathway depended on the TDP-43 state -monomeric/oligomeric, RNA-bound/-unbound- and the subcellular environment -nucleus/cytoplasm. Our work unravels the origins of heterogeneous pathological species occurring in TDP-43 proteinopathies.

cell biology↗

bioRxiv2021

Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1,2, which involve human-specific mechanisms3-6 that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors7. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs8, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids9. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.

neuroscience↗

bioRxiv2020

Synaptic accumulation of FUS triggers age-dependent misregulation of inhibitory synapses in ALS-FUS mice

FUS is a primarily nuclear RNA-binding protein with important roles in RNA processing and transport. FUS mutations disrupting its nuclear localization characterize a subset of amyotrophic lateral sclerosis (ALS-FUS) patients, through an unidentified pathological mechanism. FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Here, we used super-resolution imaging to determine the physiological localization of extranuclear, neuronal FUS and found it predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosome preparations, we identified synaptic RNA targets of FUS that are associated with synapse organization and plasticity. Synaptic FUS was significantly increased in a knock-in mouse model of ALS-FUS, at presymptomatic stages, accompanied by alterations in density and size of GABAergic synapses. RNA-seq of synaptoneurosomes highlighted age-dependent dysregulation of glutamatergic and GABAergic synapses. Our study indicates that FUS accumulation at the synapse in early stages of ALS-FUS results in synaptic impairment, potentially representing an initial trigger of neurodegeneration.

neuroscience↗