Research Projects

Dengue virus (DENV) infection is a global health threat, with severe cases marked by thrombocytopenia that predisposes patients to bleeding and shock. Platelet production depends on megakaryocyte (MK) differentiation, regulated by transcriptional and post-transcriptional networks. Emerging evidence implicates long non-coding RNAs (lncRNAs) and the spliceosome in megakaryopoiesis, yet their roles in DENV-induced platelet deficits remain unexplored. This proposal investigates how DENV manipulates the lncRNA interactome and host splicing in PMA-induced K562 cells to elucidate thrombocytopenia mechanisms and identify biomarkers and therapeutic targets. Rationale. Platelet biogenesis requires endomitosis and proplatelet formation coordinated by factors like GATA1 and NF-E2. lncRNAs regulate these processes by scaffolding chromatin modifiers and modulating mRNA stability. Meanwhile, DENV NS5 binds U5 snRNP components, causing widespread splicing errors. Since platelets inherit RNA from MKs, DENV-driven lncRNA and splicing dysregulation likely impair differentiation and platelet output. Objectives. 1. Elucidate the DENV‐Responsive lncRNA Landscape in Megakaryopoiesis. 2. Map splicing alterations induced by DENV and identify affected spliceosomal proteins. 3. Integrate lncRNA–Spliceosome Interplay for Mechanistic Insight and Biomarker Discovery. Hypothesis. DENV infection disrupts megakaryopoiesis by altering specific lncRNAs that regulate differentiation and by hijacking the spliceosome to produce aberrant isoforms of critical MK genes, jointly causing thrombocytopenia. Main Experiments. • Transcriptomics & Interactome Mapping: RNA-seq of PMA-treated K562 ± DENV; ChIRP/RIP to identify lncRNA partners. • Splicing Analysis: Use rMATS or MAJIQ to detect DENV-induced alternative splicing; confirm NS5–snRNP interactions via immunoprecipitation and mass spectrometry. • Functional Assays: Knockdown/overexpress candidate lncRNAs; assess polyploidization (flow cytometry), CD41/CD61 levels, and proplatelet formation. Apply splice-switching oligonucleotides to correct key splicing errors and evaluate phenotype rescue. • Viral Fitness: Measure DENV load by qRT-PCR and focus-forming assays in manipulated cells. • Clinical Correlation: Quantify candidate lncRNAs and splice isoforms in platelets from dengue patients and correlate with platelet counts and severity. Significance. This work will establish a mechanistic link between lncRNA dysregulation, splicing perturbation, and DENV-induced thrombocytopenia. Outcomes include: • Fundamental insight into non-coding RNA and splicing control of hematopoietic differentiation under viral stress. • Biomarkers for early prediction of severe dengue based on lncRNA and splice isoform signatures. • Therapeutic avenues using oligonucleotides to restore normal lncRNA function or correct splicing defects, with broader relevance to other hematological and viral diseases. By integrating transcriptomic, interactomic, and functional strategies, the project aims to transform understanding of platelet biogenesis in dengue and enable targeted diagnostics and interventions to reduce disease burden.