Research Projects

Maintenance of genome integrity is critical for cellular homeostasis and human health. While the role of RNA Polymerase II (RNAPII) termination and its associated factors in transcription stress and genome instability is well-established. RNAPIII, responsible for transcribing essential small RNAs such as tRNAs and 5S rRNA, remains poorly understood in this context. Recent evidence suggests that Sen1, a conserved helicase (human homolog Senataxin/SETX), plays a critical role in RNAPIII transcription termination and R-loop resolution. In humans, mutations in RNAPIII components are linked to neurological disorders such as hypomyelinating leukodystrophy, while mutations in SETX are causative of severe neurodegenerative diseases including Ataxia with Oculomotor Apraxia type 2 (AOA2) and Amyotrophic Lateral Sclerosis type 4 (ALS4). We hypothesize that dysfunction of SETX impairs RNAPIII transcription termination, leading to the accumulation of R-loops (three-stranded nucleic acid structures), DNA damage, and ultimately, the onset of neurodegenerative diseases such as AOA2 and ALS4. This project seeks to elucidate the molecular mechanisms by which Sen1/SETX regulates RNAPIII transcription and genome integrity, using the genetically tractable yeast S. pombe and a disease-relevant rat model system. Supported by strong preliminary data, including the synthetic lethality of Δsen1 strains with RNAPIII termination-defective mutants and AOA2-linked SETX mutants introduced into S.pombe’s Sen1, impair the termination activity of RNAPIII. The proposal is structured around the following four objectives: 1. Characterization of AOA2-linked SETX mutations in yeast Sen1 and their functional impact on RNAPIII termination. 2. Analysis of genome instability and R-loop accumulation resulting from defective RNAPIII termination using DRIP-seq, γH2AX markers, and RNaseH1 rescue. 3. Exploration of Sen1, RNAPIII, and replisome interactions under transcriptional stress to understand Sen1’s role in resolving transcription-replication conflicts. 4. Modeling SETX–RNAPIII dysfunction in a rat system using siRNA-mediated knockdown, behavioral assays, and molecular analyses to investigate neurodegenerative phenotypes. By integrating classical yeast genetics, cutting-edge sequencing technologies, and mammalian neurobiology, this interdisciplinary project will address a major gap in our understanding of RNAPIII regulation and its role in genome maintenance. The outcomes are expected to 1) Uncover fundamental mechanisms linking transcription termination of RNAPIII and genome stability, 2) Provide mechanistic insights into SETX-associated neurodegeneration, and 3) Open window to identify novel biomarkers or therapeutic targets for diseases such as AOA2 and ALS4. Given the lack of such focused research in India, this project also aims to establish national leadership in RNAPIII biology and neurodegenerative disease modeling. This aligns closely with the goals of ANRF to foster innovation and address pressing biomedical challenges.