In partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Bioinformatics
in the School of Biological Sciences

Deepali L. Kundnani

Defends her thesis:
Distribution and features of ribomes, genome-wide embedded ribonucleotides in eukaryotic cells

Time: Monday June 23, 2025, 11:00 AM Eastern Time (US and Canada)
Hybrid: EBB CHOA 1005
Join Zoom Meeting: https://gatech.zoom.us/j/3168670242?omn=93958825468 
Thesis advisor:
Dr. Francesca Storici, School of Biological Sciences, Georgia Institute of Technology

Committee Members:
Dr. Greg Gibson, School of Biological Sciences, Georgia Institute of Technology

Dr. Mark Borodovsky, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology

Dr. Yuhong Fan, School of Biological Sciences, Georgia Institute of Technology

Dr. Nataša Jonoska, Department of Mathematics & Statistics, University of South Florida

SUMMARY
Ribonucleoside monophosphates (rNMPs) are units of RNA frequently embedded in genomic DNA by DNA polymerases across species, at a rate that makes rNMPs the most abundant non-canonical DNA alteration. The removal of these embedded rNMPs is primarily mediated by Ribonuclease H2 (RNase H2), and defects in this enzyme are directly linked to Aicardi-Goutières syndrome (AGS), a rare neuroinflammatory disorder that severely impacts the brain and immune system. Over half of AGS patients harbor mutations in one of the three RNase H2 subunits. Despite significant advances in rNMP mapping technologies since 2015, the distribution, patterns, and functions of rNMPs in the human nuclear genome remain poorly characterized, and it is largely unknown how AGS-associated mutations influence these features. 
In this study, we systematically investigate the genome-wide landscape of rNMP incorporation, referred to as the “ribome”, in AGS-orthologous mutants generated in yeast, as well as in multiple human cell types. Focusing on two of the most frequently studied AGS mutations, RNASEH2A-G37S and RNASEH2C-R69W (modeled in yeast as rnh201-G42S and rnh203-K46W, respectively), we uncover distinct features of DNA-embedded ribonucleotides at single-nucleotide resolution. Our results show that the rnh201-G42S mutant exhibits a marked increase in rNMP embedment, mirroring the phenotype of a complete rnh201 deletion, while both AGS-orthologous mutants display elevated rC content and unique sequence contexts surrounding rNMPs. Furthermore, comparison with a yeast RNase H2B mutant that disrupts binding to the sliding-clamp proliferating cell nuclear antigen (PCNA) (rnh202-pip) suggests that the rnh203-K46W mutation may similarly impair the enzyme’s interaction with PCNA, potentially affecting DNA replication and repair. In human ribomes, rNMPs are enriched at transcription start sites (TSSs) and CpG islands, with conserved ribonucleotide-enriched zones (REZs) co-localizing with these genomic features. Notably, rNMP enrichment near TSSs correlates positively with gene expression in both wild-type and RNase H2 knockout cells. However, knockout cells exhibit pronounced retention of embedded rG and a strand-biased decrease in template rC near TSSs, both of which intensify with higher gene expression. We also discovered conserved rNMP patterns in telomeres across diverse human cell types. Collectively, these findings establish DNA-embedded rNMPs as a novel form of epigenetic marking, shedding light on the molecular consequences of AGS mutations, the regulatory roles of rNMPs in the human genome, and their potential contributions to aging and disease.