Studying R-loops in development and disease

Abstract

Studying R-loops in development and disease Hyo Jung Sim1, Keun Yeong Kwon1, Taejoon Kwon2, and Tae Joo Park1,* 1 Department of Biological Sciences, College of Information-Bio Convergence, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea, 2 Department of Biomedical Engineering, College of Information-Bio Convergence, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea The formation of R-loops, which involves the invasion of the nascent RNA into the DNA duplex, has long been associated with genomic instability and implicated in various pathogenesis. However, recent studies reported that the R-loops also participated in physiological roles, such as mitochondrial DNA replication, and regulating transcription. We assumed that these physiological R-loop may be involved in essential cellular processes. For this reason, we investigated the participation of R-loops in developmental processes, focusing on the gastrula stage of Xenopus embryos, in which the maternal-zygotic transition occurs. The CUT&TAG (Cleavage Under Targets & Tagmentation) technique was employed to analyze R-loop dynamics during gastrulation and to explore their potential relationship with physiological processes. Surprisingly, we observed a substantial presence of R-loops in telomere regions, suggesting a potential connection between R-loops and telomere elongation. Furthermore, we hypothesized that the long non-coding RNA TERRA (Telomeric Repeat-containing RNA) might play a role in this telomere-associated R-loop formation. In addition to elucidating the physiological roles of R-loops, we aimed to determine pathological R-loops. We first employed the CUT&TAG technique and analyzed a DDX41 mutant cell line, which lacks the helicase activity necessary for R-loop resolution and is associated with leukemia. Remarkably, our analysis of the DDX41 mutant cell line revealed a set of genes associated with leukemia. Based on this insight, we are developing a leukemia disease model using Xenopus and are in the process of validating it. Furthermore, we utilized an egg extract system to investigate R-loop binding proteins, aiming to identify novel R-loop binding proteins and specific proteins that bind to physiological or pathological R-loops. We are looking forward to this study will uncover the involvement of R-loops in developmental processes. Additionally, we expect that our investigation into the determining R-loops as physiological or pathological can provide crucial insights for future studies in this field.