Biology subjects

Jagannathan, S.

Publications and source records attributed to Jagannathan, S..

13 recordsLinked to original sources

bioRxiv2026

Unveiling the Hidden Rules: Enhancing NMD Prediction for Protein-Truncating Variants

Nonsense-mediated decay (NMD) is a conserved RNA quality-control pathway that degrades transcripts containing premature termination codons. Because roughly a third of pathogenic variants in ClinVar can lead to truncated protein synthesis, predicting whether such transcripts undergo NMD is central to interpreting variant effects, yet the canonical 50-55 nucleotide rule explains only about half of observed outcome variability. Using paired whole-genome and RNA-sequencing from 10,306 individual samples in the Trans-Omics for Precision Medicine (TOPMed) program, we quantified NMD efficiency for 5,749 germline truncating variants via allele-specific expression and trained a gradient-boosting classifier, TrunCat, that distinguished NMD-sensitive from NMD-escape transcripts with [~]78% ROC-AUC (Receiver Operating Characteristic - Area Under the Curve). A reduced model using the ten features with the highest mean SHAP (SHapley Additive exPlanations) value as a measure of each features average contribution to predictions nearly matched this performance. Applied across large variant databases and a rare-disease cohort, the model produced NMD outcome predictions, with variants of uncertain significance showing higher predicted escape than pathogenic ones. This framework confirms the canonical rule, identifies non-canonical determinants, and offers a scalable resource for interpreting protein-truncating variants.

genomics↗

bioRxiv2026

Multi-feature Classification to Improve Colorimetric Loop-Mediated Isothermal Amplification Fidelity

Loop-mediated isothermal amplification (LAMP) is a cost-effective and portable assay technique for performing nucleic acid-based diagnostics in the field whose adoption is hindered by design and reproducibility issues. This is due to a complex primer design process that fine-tunes parameters across 6-8 binding regions. The likelihood of assay success depends on satisfying thermodynamic and secondary structure constraints while maintaining target specificity and avoiding overlaps between multiple primers. Software such as the NEB(R) LAMP Primer Design Tool, PREMIER Biosoft LAMP Designer, Primer3, PCR Signature Erosion Tool (PSET), and PrimerExplorer enable automation of this task for researchers. However, in our experience, these programs can sometimes yield inconsistent results in laboratory testing. Here, we approached the issue by comparing and training multiple machine learning (ML) models on primer sets targeting various organisms from working assays and failing ones to determine significant features and improve predictions prior to ordering primer sets. A literature review produced an initial list of primer sets (n=116), which were then filtered down based on reference template availability to discern their FIP/BIP components (F2/F1c and B1c/B2). The final training set (n=109) included sequence and thermodynamic features derived from primers collected from the review (n=74) and those designed in-house with PSET (n=35). Failing assays were difficult to obtain from the publications, so we provided our own (n=23). Using WEKA Experimenter, models were created based on decision tree and Bayesian learning algorithms using an experimental scheme that performed a parameter grid search, seeded replicates, feature selection, and cross-validation while avoiding data-leakage and outputting logs for model comparison, feature analysis, and overfit assessment. Notably, thermodynamic features associated with the F1c and B1c primers consistently appeared in the top ranks according to consensus between information gain, class-correlation, and model-based feature ranking. For classification, the NaiveBayes algorithm had a TP and TN rate of 0.90 ({+/-} 0.02) and 0.73 ({+/-} 0.05) while achieving Cohens kappa coefficient and F-score values of 0.61 ({+/-} 0.06) and 0.91 ({+/-} 0.01). This work highlights how a practical model was built from a small, imbalanced training set incorporating negative research results, of which more are needed to improve generalization and refine parameters critical to assay success.

bioinformatics↗

bioRxiv2025

Genomic stop codon scanning reveals quantitative principles of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) degrades transcripts containing premature termination codons (PTCs), critically shaping the disease outcomes of protein-truncating variants. While existing NMD rules categorize PTCs as NMD-triggering or NMD-evading, they cannot quantitatively predict the degree of NMD activity for a given endogenous PTC variant. To provide quantitative insight into NMD, we used saturation genome editing (SGE) to systematically introduce all possible PTCs (TAA, TAG, TGA) at every codon position spanning the first, penultimate, and three internal exons of the Lamin A/C (LMNA) gene. Combining targeted sequencing with NMD inhibition, we measured mRNA expression and NMD activity for 722 PTCs and 211 single nucleotide variants (SNVs). Our data validate known positional trends in NMD activity but reveal unexpected complexity. In the penultimate exon, the PTC position effect extends beyond the binary 50-55 nucleotides (nt) rule, revealing a quantitative relationship between the PTC-EJC distance and NMD activity. At the 5 end, NMD is completely absent in the first 21 codons of LMNA, followed by sharp activation over the next 4 codons. Both patterns, at the 5 end and in the penultimate exon, are unexplained by current models. Finally, internal exons show robust NMD with outliers consistently mapping to predicted readthrough-permissive sequence contexts, including the conserved readthrough promoting TGA-CT motif. This comprehensive dataset provides an unprecedented resource for understanding the quantitative impact of PTC position and sequence context on NMD, with direct implications for the clinical interpretation of nonsense variants in the human population.

genomics↗

bioRxiv2025

Splice-switching antisense oligonucleotides correct cryptic exon inclusion and restore SDCCAG8 protein in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a ciliopathy often associated with progressive blindness and obesity. A patient presenting with BBS was discovered to have two mutations within 55bp of each other in intron 7 of SDCCAG8 (BBS16). One of the biallelic mutations, c.740+356C>T, causes inclusion of cryptic exon(s) containing premature termination codons, while c.740+301G>A has not been characterized. We hypothesized that antisense oligonucleotides (ASOs) complementary to the patients mutations or to the cryptic exon splice sites would correct the splicing of SDCCAG8 between exons 7 and 8 to prevent cryptic exon inclusion and restore SDCCAG8 expression. We systematically screened 20nt-long ASOs tiled across each mutation and ASOs targeting the 3' splice sites of the cryptic exons in patient-derived fibroblasts, using RT-PCR assays to assess exon 7 and 8 splicing. We identified one ASO for each mutation and a cryptic exon-targeting ASO that restored the splicing pattern to that observed in an unaffected cell line. Lead ASOs were further investigated through RT-PCR, RNA sequencing, and western blotting to confirm ASO-mediated restoration of wild-type transcript and protein. Notably, ASO 20, which targets the cryptic exon 7a/7a' splice site rather than patient-specific mutations, achieved the greatest rescue effect, increasing exon 7-8 splicing from 0% to an average of 26% and restoring SDCCAG8 protein from undetectable levels to approximately 40% of wild-type expression. This mutation-agnostic approach could benefit multiple patients with cryptic exon inclusion in this region of SDCCAG8, expanding therapeutic impact beyond traditional N-of-1 ASO strategies. These findings establish a molecular foundation for clinical development of ASO therapy for BBS caused by SDCCAG8 splicing defects.

genetics↗

bioRxiv2025

Advancements in Vaccine Development : A comprehensive design of a Multi-Epitopic Immunodominant Peptide Vaccine Targeting Kyasanur Forest Disease via Reverse Vaccinology

Kyasanur Forest Disease (KFD), caused by the Kyasanur Forest Disease Virus (KFDV), is a tick-borne hemorrhagic fever virus first identified in Karnataka, South India, in 1957. The rising incidence of cases is alarming, particularly due to the potential spread of the disease to neighboring states. There presently exists no particular treatment, and the Chick embryo fibroblast (CEF) vaccine only provides temporary immunity and necessitates many doses, which results in poor immunisation rates among at-risk groups. This situation highlights the urgent need for a highly effective and user-friendly vaccine. Given that KFDV is classified as a BSL-4 pathogen, this study employs advanced immuno-informatics tools to design a safer vaccine construct. Promising results have identified the E protein of KFDV as an immunodominant target that can stimulate robust immune responses, including the production of neutralizing antibodies. The vaccine design utilized various in silico tools to predict CTL, HTL and B-cell epitopes from the E protein, ensuring powerful immunological reactions. Quality attributes such as antigenicity, allergenicity, toxicity, and immunogenicity were assessed using specialized tools. A total of 16 epitopes (5 CTL, 3 HTL, and 8 B-cell) were identified, all exhibiting desirable properties like non-toxicity, non-allergenicity, high antigenic and immunogenic value. These epitopes were linked to form the PKFDVac-I vaccine, consisting of 279 amino acids with a MW of 29,162.32 Daltons. The constructed vaccine demonstrated stability and a robust immune response when docked with the TLR-4 receptor, indicating strong immunogenic potential. This multi-epitope peptide vaccine candidate developed via user-friendly and safer bio-informative tools represents a significant advancement in effective immunization strategies against KFDV infection, paving the way for future vaccine development efforts targeting other emerging infectious diseases. In conclusion, the integration of diverse epitopes into a cohesive vaccine prototype demonstrates a promising avenue for custom synthesis and application in immunization strategies. Further validation through in vitro and in vivo studies is essential to authorize the effectiveness of the designed vaccine construct.

microbiology↗

bioRxiv2024

Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). Despite its clinical importance, the factors controlling transcript-specific and context-dependent variation in NMD activity remain poorly understood. Through analysis of human genetic datasets, we discovered that the amino acid preceding the PTC strongly influences NMD activity. Notably, glycine codons promote robust NMD efficiency and show striking enrichment before PTCs but depletion before normal termination codons (NTCs). This glycine-PTC enrichment is particularly pronounced in genes tolerant to loss-of-function variants, suggesting evolutionary selection or neutrality conferred by efficient elimination of truncated proteins from non-essential genes. Using biochemical assays and massively parallel reporter analysis, we demonstrated that the peptide release rate during translation termination varies substantially with the identity of the preceding amino acid and serves as the primary determinant of NMD activity. We propose a "window of opportunity" model where translation termination kinetics modulate NMD efficiency. By revealing how sequence context shapes NMD activity through translation termination dynamics, our findings provide a mechanistic framework for improved clinical interpretation of nonsense variants.

molecular biology↗

bioRxiv2023

A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

molecular biology↗

bioRxiv2023

Effects of alertness on perceptual detection and discrimination

The level of alertness fluctuates throughout the day, exerting modulatory effects on human cognitive processes at any moment. However, our knowledge of how alertness level interacts with specific cognitive demands and perceptual rules of the task is still limited. Here we use perceptual decision-making paradigms to understand how alertness modulates the detection of a stimulus and the capacity to discriminate one stimulus from another. We analyzed data from four different experiments (113 participants in total): 1 - auditory masking detection; 2 - sensorimotor detection; 3 - auditory spatial discrimination; and 4 - auditory phoneme discrimination, and examined the performance of participants during the natural transition from awake (high alertness) to drowsy (low alertness). First, we fitted psychometric functions to the hit rates across different conditions of difficulty for EEG-defined high and low alertness metastable states, respectively. Second, we performed modelling of slope and threshold for the fitted curves as well as signal detection theory measures of perceptual sensitivity (d) and response bias (criterion). We found lower detection and discrimination sensitivity to stimuli as alertness level decreases, signalled by a shallower slope of the sigmoidal curve and a lower d, while the threshold increases slightly and equivalently across experiments during lower alertness. There was no change in the criterion to make the decision during the transition. These results suggest that reduced alertness generally decreases the quality of perceptual decision-making. Zooming in, we observed that the decrease in sensitivity measured by slope was stronger for discrimination than for detection decisions, indicating that lower alertness impairs the precision of decisions in discriminating alternatives more than identifying the presence of a stimulus around the threshold. Taken together, these results suggest that alertness has a common effect on perceptual decision-making and differentially modulates detection and discrimination decisions.

animal behavior and cognition↗

bioRxiv2021

The myopathic transcription factor DUX4 induces the production of truncated RNA-bindingproteins in human muscle cells

Nonsense-mediated RNA decay (NMD) is a surveillance mechanism that degrades both canonical and aberrant transcripts carrying premature translation termination codons. NMD is thought to have evolved to prevent the synthesis of toxic truncated proteins. However, whether global inhibition of NMD results in widespread production of truncated proteins is unknown. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD) features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Here, using a cell-based model of FSHD, we show the production of hundreds of truncated proteins from physiological NMD targets. Using ribosome profiling, we map the precise C-terminal end of these aberrant truncated proteins and find that RNA-binding proteins are especially enriched for aberrant truncations. The stabilized NMD isoform of one RNA-binding protein, SRSF3, is robustly translated to produce a stable truncated protein, which can also be detected in FSHD patient-derived myotubes. Notably, ectopic expression of truncated SRSF3 alone confers toxicity and its downregulation is cytoprotective. Our results demonstrate the genome-scale impact of NMD inhibition. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

genomics↗

bioRxiv2021

Selective recruitment of endoplasmic reticulum-targeted and cytosolic mRNAs into membrane-associated stress granules

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. The biochemical criteria for mRNA recruitment into SGs are largely unknown. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e. endoplasmic reticulum (ER)-targeted) transcripts are significantly under-represented, consistent with prior reports that ER-localized mRNAs are excluded from SGs. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis during the unfolded protein response (UPR) and report that UPR-elicited SG formation is gene selective. Combined immunofluorescence-smFISH studies demonstrated that UPR-induced mRNA granules co-localized with SG protein markers and were in close physical proximity to or directly associated with the ER membrane. mRNA recruitment into ER-associated SGs required stress-induced translational inhibition, though translational inhibition was not solely predictive of mRNA accumulation in SGs. SG formation in response to UPR activation or arsenite addition was blocked by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. These data demonstrate that ER-targeted mRNAs can be recruited into SGs and identify the ER as a subcellular site of SG assembly. On the basis of the transcriptional inhibitor studies, we propose that newly transcribed mRNAs undergoing nuclear export during conditions of suppressed translation initiation are key substrates for SG biogenesis.

cell biology↗

bioRxiv2020

Information flow, cell types and stereotypy in a full olfactory connectome

The hemibrain connectome provides large scale connectivity and morphology information for the majority of the central brain of Drosophila melanogaster. Using this data set, we provide a complete description of the Drosophila olfactory system, covering all first, second and lateral horn-associated third-order neurons. We develop a generally applicable strategy to extract information flow and layered organisation from connectome graphs, mapping olfactory input to descending interneurons. This identifies a range of motifs including highly lateralised circuits in the antennal lobe and patterns of convergence downstream of the mushroom body and lateral horn. Leveraging a second data set we provide a first quantitative assessment of inter - versus intra-individual stereotypy. Comparing neurons across two brains (three hemispheres) reveals striking similarity in neuronal morphology across brains. Connectivity correlates with morphology and neurons of the same morphological type show similar connection variability within the same brain as across two brains.

neuroscience↗

bioRxiv2019

Co-Stimulation-Induced AP-1 Activity is Required for Chromatin Opening During T Cell Activation

Activation of T cells is dependent on organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 hours of activation. These newly open regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at more than 70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation, was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1-dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.

immunology↗

bioRxiv2018

Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD

DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). While DUX4s transcriptional activity has been extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-induced pathology.

genomics↗