Transcript RNA-mediated mechanisms in end-joining repair of DNA double-strand breaks

About This Grant

Project Summary Double-strand breaks (DSBs) in DNA represent one of the most severe threats to genomic integrity, with improper repair leading to mutations, chromosomal translocations, and oncogenic transformation. Cells have evolved robust pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), to repair DSBs. While much is known about protein-mediated DSB repair mechanisms, the role of transcript RNA in modulating repair remains poorly understood. Yet, the potential contribution of RNA molecules, particularly transcript RNA with sequence complementarity to DNA ends, remains largely unexplored. Recent discoveries from the PI’s laboratory demonstrate that RNA transcripts can bridge broken DNA ends and facilitate repair via NHEJ and MMEJ in both human and yeast systems, suggesting a conserved and fundamental role for RNA in genome maintenance. This project seeks to systematically uncover the mechanisms, regulatory proteins, and biological consequences of transcript RNA involvement in DSB repair across multiple contexts in human cells. Aim 1 establishes isogenic human embryonic kidney (HEK293T) and diploid retinal pigment epithelial (RPE-1) cell lines expressing spliced or non-spliced RNAs from the same adeno-associated virus integration site 1 (AAVS1) safe harbor locus and investigates how these transcripts modulate end-joining repair (NHEJ and MMEJ) of chromosomal DSBs induced by CRISPR/Cas9, followed by deep sequencing and bioinformatics analysis. Aim 2 identifies the key proteins and molecular processes involved in RNA-mediated end-joining repair in human cells. This goal will be achieved by inhibiting core NHEJ and MMEJ factors using chemical inhibitors and siRNAs, and by selectively reducing nascent spliced and non-spliced RNA transcripts using CRISPRi. These perturbations will define how individual proteins and transcript RNAs contribute to the efficiency, fidelity, and pathway choice of RNA-mediated end joining. Aim 3 examines whether transcript RNA differentially modulates DSB repair in exonic and intronic sequences of endogenous human genes in both HEK293T and RPE-1 cells, including breaks induced by CRISPR/Cas9 and mutagens, to understand RNA’s role in maintaining genome integrity. The significance of this work lies in establishing transcript RNA as a previously unrecognized regulator of DSB repair pathways, revealing new dimensions of genome stability control and potential therapeutic targets. The results could provide insights to improve genome editing technologies, inform strategies to reduce mutagenesis and carcinogenesis, and contribute fundamentally to understanding how RNA functions extend beyond traditional regulatory roles to directly safeguarding the genome. The outcomes will also lay groundwork for exploring RNA-mediated DNA repair in stem cells, non-dividing cells, aging models, and disease states, potentially transforming approaches to cancer prevention and therapy.