Deepali L Kundnani, Yeunsoo Lee, Taehwan Yang, Tyler P Warner, Francisco Martinez-Figueroa, Tejasvi Channagiri, Penghao Xu, Ryan A Eckerty, Mo Sun, Supreet Randhawa, Youngkyu Jeon, Stefania Marsili, Gary Newnam, Yilin Lu, Miharu Yasumuro, Ashlesha Gogate, Vivian S Park, Sijia Tao, Justin A Ling, Ryohei Chiba, Hiroyoshi Matsumura, Ryo Uehara, Jonathan M Fogg, Lynn Zechiedrich, Raymond F Schinazi, Zachary F Pursell, Abdulmelik Mohammed, Patricia L Opresko, Bret D Freudenthal, Baek Kim, Soojin V Yi, Nataša Jonoska, Francesca Storici
Ribonucleoside monophosphates (rNMPs) are the most abundant non-canonical nucleotides in DNA, yet their distribution and function in the human nuclear genome remain unclear. We present high-resolution maps of ∼1 million rNMPs per genome across diverse human cells, revealing a non-random nuclear "ribome," the genome-wide landscape of embedded rNMPs, enriched in GC-rich regions, regulatory elements, and telomeres. Ribonucleotide-enriched zones (REZs) cluster near transcription start sites (TSSs), coincide with C-phosphate-G (CpG) islands, R-loops, and G4 structures, and scale with gene expression. Ribonuclease (RNase) H2 deficiency increases rGMP levels and is associated with topoisomerase 1 (Top1)-dependent, strand-biased rNMP enrichment near TSSs, while Top1 depletion further enhances rGMP accumulation. RNase H2-mediated nicking at rNMPs alters DNA supercoiling in vitro, and RNH2A-deficient cells show altered supercoiling at rNMP-enriched TSSs. Our findings identify embedded rNMPs as epigenetic modulators of DNA supercoiling linked to DNA sequence and transcription, revealing a connection between ribonucleotide processing and transcription-associated DNA topology in human cells.