Francesca Storici

Ribonucleoside monophosphates (rNMPs) are abundant in DNA, but their distribution and function in human nuclear genomes remain unknown. Here, we mapped nearly one million rNMPs per genome across diverse human cell types, defining a nuclear “ ribome ” with non-random distribution patterns. rNMPs are enriched in C/G-rich sequences, epigenetically marked regions, and telomeres. Conserved ribonucleotide-enriched zones (REZs) overlap with CpG islands and R-loops. rNMP concentration near transcription start sites (TSSs) correlates positively with gene expression. Wild-type cells display a broader gene-expression range than ribonuclease H2A (RNH2A) knockouts, in which loss of rNMP cleavage causes pronounced retention of embedded rG and strand-biased rC near TSSs, both increasing with gene expression. These findings establish DNA-embedded rNMPs as a novel epigenetic mark that modulates human gene expression.

Genome integrity is constantly challenged by the incorporation of ribonucleotides (rNMPs) during DNA synthesis. Covalently linked single and several consecutive rNMPs occur in the genome of a number of organisms. They are mainly introduced by DNA polymerases during DNA replication and repair. In general, cells evolved ribonucleases H (RNases H) specialized in the removal of rNMPs from DNA to avoid any detrimental consequences on genome stability. Here, we describe the involvement of types 1 and/or 2 RNases H in processing embedded rNMPs in the genome of two archaeal species Haloferax volcanii and Thermococcus barophilus . Using combined approaches that include alkaline DNA fragmentation, high-throughput ribose-seq DNA sequencing and nucleotide pool quantification, the distribution, identity, level and sequence context of genomic rNMPs are reported and discussed regards to the intracellular balances of dNTPs and rNTPs. Our results confirm the predominant role of type 2 RNase H in the removal of genomic rNMPs. They also reveal rNMP-base compositions, densities, locations, and variations of surrounding bases at rNMP-embedment for each mutant. The cellular roles of the different RNases H in processing rNMPs in the genome of Archaea are discussed.

Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.

Motivation: Ribonucleoside monophosphates (rNMPs) are the most abundant non-standard nucleotides embedded in genomic DNA. If the presence of rNMP in DNA cannot be controlled, it can lead to genome instability. The actual regulatory functions of rNMPs in DNA remain mainly unknown. Considering the association between rNMP embedment and various diseases and cancer, the phenomenon of rNMP embedment in DNA has become a prominent area of research in recent years. Results: We introduce the rNMPID database, which is the first database revealing rNMP-embedment characteristics, strand bias, and preferred incorporation patterns in the genomic DNA of samples from bacterial to human cells of different genetic backgrounds. The rNMPID database uses datasets generated by different rNMP-mapping techniques. It provides the researchers with a solid foundation to explore the features of rNMP embedded in the genomic DNA of multiple sources, and their association with cellular functions, and, in future, disease. It also significantly benefits researchers in the fields of genetics and genomics who aim to integrate their studies with the rNMP-embedment data. Availability and implementation: rNMPID is freely accessible on the web at https://www.rnmpid.org.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Motivation: Ribonucleoside monophosphates (rNMPs) are the most abundant non-standard nucleotides embedded in genomic DNA. If the presence of rNMP in DNA cannot be controlled, it can lead to genome instability. The actual positive functions of rNMPs in DNA remain mainly unknown. Considering the association between rNMPs embedment and various diseases and cancer, the phenomenon of rNMPs embedment in DNA has become a prominent area of research in recent years. Results: We introduce the rNMPID database, which is the first database revealing rNMP-embedment characteristics, strand bias, and preferred incorporation patterns in the genomic DNA of samples from bacterial to human cells of different genetic backgrounds. The rNMPID database uses datasets generated by different rNMP-mapping techniques. It provides the researchers with a solid foundation to explore the features of rNMPs embedded in the genomic DNA of multiple sources, and their association with cellular functions, and, in future, disease. It also significantly benefits researchers in the fields of genetics and genomics who aim to integrate their studies with the rNMP-embedment data. Availability: rNMPID is freely accessible on the web at https://www.rnmpid.org . Contact: xph6113@gmail.com or storici@gatech.edu

SUMMARY: Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS-ortholog mutations in Saccharomyces cerevisiae : rnh201 -G42S and rnh203 -K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these yeast AGS-ortholog mutants. We found higher rNMP incorporation in the nuclear genome of rnh201 -G42S than in wild-type and rnh203- K46W-mutant cells, and an elevated rCMP content in both mutants. Moreover, we uncovered unique rNMP patterns in each mutant, highlighting a differential activity of the AGS mutants towards rNMPs embedded on the leading or on the lagging strand of DNA replication. This study guides future research on rNMP characteristics in human genomic samples carrying AGS mutations.

Summary: Double-strand breaks (DSBs) in DNA are challenging lesions to repair. Human cells employ at least three DSB repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ) ^1,2 . In contrast to HR, NHEJ and MMEJ do not utilize a DNA template molecule to recover damaged and/or lost nucleotides ² . NHEJ directly ligates broken DNA ends, while MMEJ exploits the alignment of short microhomologies on the DSB sides and is associated with deletions of the sequence between the microhomologies ^3,4 . It is unknown whether and to what extent a transcript RNA has a direct role in DSB-repair mechanisms in mammalian cells. Here, we show that both coding and non-coding transcript RNA facilitates DSB repair in a sequence-specific manner in human cells. Depending on its sequence complementarity with the broken DNA ends, the transcript RNA could promote the repair of a DSB or gap in its DNA gene via NHEJ or MMEJ, or mediate RNA-templated repair. The transcript RNA influences DSB repair by NHEJ and MMEJ even when the transcription level is low. The results demonstrate an unexpected role of transcript RNA in directing the way DSBs are repaired in human cells and maintaining genome stability.