ICGEB Alumna Francesca Storici is a multi-awarded HHMI Faculty Scholar, GRA Distinguished Cancer Scientist and Professor at the Georgia Institute of Technology in Atlanta, USA. In Trieste in July to attend the Arturo Falaschi Conference, she spoke to us about her research and about her time as PhD student at ICGEB.
Prof. Storici is an active, high-flying member of the ICGEB Alumni Association. With a view to consolidating this network, she is spearheading the initiative to introduce an Alumni Mentorship Programme to connect senior and junior Alumni through online mentorship and in-person mentoring in Alumni-headed labs around the world.
During her PhD at ICGEB, Francesca discovered a preference for working with eukaryote cells and yeast plasmids and also had the opportunity to directly follow and learn from the creation of one of the first yeast genome databases, by manually sequencing yeast plasmids. At present, much of these processes have been automated, thus her work heavily relies on bioinformatics for analysing the high amount of data. Her lab works on elucidating the mechanisms of DNA recombination and repair and developing approaches for genetic engineering and gene targeting. A major focus of her research is understanding the relationship between RNA and DNA in genome stability/instability and DNA modification.
Prof. Francesca Storici enjoys returning to her native Trieste and reconnecting with colleagues. In the context of the ICGEB International Seminar Programme, she presented a seminar on her latest findings on the tight relationship between RNA and DNA in the genome. We are delighted to publish the ensuing article, below.
The tight relationship between RNA and DNA in the genome
by Francesca Storici
School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA
Over the last decade, it has become apparent that RNA and DNA have a very tight relationship with each other. However, features, molecular mechanisms, and significance of such interconnected relationships remain mostly obscure. Here, I present work from my group that i) has uncovered the capacity of RNA to directly interact with DNA in the process of repairing DNA damage, and ii) has led to the development of new molecular and bioinformatics approaches to study and characterize the features of RNA nucleotides embedded in genomic DNA of cells.
Double-strand break (DSB) repair by transcript RNA. A DNA DSB resulting from exogenous DNA damaging agents or faulty DNA metabolism is an extremely harmful type of DNA lesion, which causes mutations or cell death, and can lead to cancer and other genetic disorders (Heyer et al., 2010). To minimize these effects, cells use DNA repair pathways, like homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) (Heyer et al., 2010; Paques and Haber, 1999; Symington and Gautier, 2011). Only HR generally performs DSB repair in an error-free manner (Heyer et al., 2010; Paques and Haber, 1999; Symington and Gautier, 2011) by retrieving the missing information from a DNA homologous template molecule. NHEJ re-ligates the broken ends, with no need of a homologous DNA template. NHEJ is often mutagenic leading in small insertions or deletions at the site of the DSB (Hefferin and Tomkinson, 2005; Symington and Gautier, 2011). NHEJ can also generate gross rearrangements by joining the wrong DSB ends (Symington and Gautier, 2011). MMEJ requires short end degradation that exposes few nucleotide microhomologies between the two sides of the DSB that can align with each other resulting in the deletion of the sequence between the microhomologies (Seol et al., 2018). When cells are in S or G2 phase of the cell cycle, HR utilizes sister chromatids as the source of identical (or nearly identical) DNA templates for DSB repair. In non-dividing cells that are in the G0 or G1 phase of the cell cycle, no sister chromatids are available, thus there is no alternative but to repair DSBs via the mutagenic NEHJ or MMEJ pathway, or by invading a homologous but not identical sequence, which can lead to mutations, rearrangements or loss of heterozygosity (Heyer et al., 2010).
Can RNA have a direct role in DSB repair? Because a great part of genomic DNA of cells is transcribed into RNA, we have hypothesized that the flow of genetic information from DNA to RNA can be reversed in a more general manner than anticipated beyond the special cases of reverse transcription of retroelements (Meers et al., 2016). The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells synthetize abundant RNA, and RNA has a wide range of functions, still much is unknown about the role of RNA in the maintenance of genome stability, DNA repair, and repair of DSBs. We have examined whether the flow of genetic information from RNA to DNA can occur during repair of DSBs.
RNA is transcribed from DNA; thus, if there is a DSB in a transcribed DNA region, the RNA transcript could in principle serve as a template for the repair of the DSB because it contains the same genetic information of that DNA region. Moreover, during the process of transcription, RNA is physically nearby DNA, and RNA/DNA hybrids can form frequently (Waldron, 2017). Still very little is known about the capacity of RNA to directly participate in DNA repair. In fact, better understanding is needed about the relationship between RNA and DNA in genome stability. While most of mammalian genomic DNA is transcribed into RNA, only a small percentage of transcribed human genome has an identified function. Moreover, understanding mechanisms of RNA-mediated DSB repair could be relevant to develop new tools and improve existing tools for genome editing.
Our recent work in budding yeast Saccharomyces cerevisiae has demonstrated that genetic information carried on transcript RNA can flow back to chromosomal DNA either directly, or indirectly via the conversion of RNA into a DNA copy (cDNA) intermediate if RNA or cDNA recombines with DNA in the process of DSB repair (Keskin et al., 2014). We found that deficiency of ribonuclease H (RNase H) proteins, which cleave RNA in a hybrid with DNA (Cerritelli and Crouch, 2009), allows detection of DSB repair by transcript RNA (Keskin et al., 2014). Thus, even G0 or G1 cells could potentially repair a DSB using a perfectly identical template provided by RNA. However, the mechanisms and circumstances in which transcript RNA recombines with homologous DNA are mostly uncharacterized.
Our studies revealed that RNA-templated DSB repair requires abundant transcription, is independent from the reverse transcriptase function of the yeast retrotransposon, and utilizes the DNA polymerase “zeta” or “delta” (Meers et al., 2020). Moreover, we found that the recombination protein Rad52 is a key factor, and the activity of mismatch repair is needed most likely to correct the mutations introduced by DNA Pol “zeta”. DSB repair by cDNA instead is fully dependent on the reverse transcriptase function of the yeast retrotransposons, requires Rad52, as well as clippase function by the Rad1/Rad10 proteins (Meers et al., 2020) (Figure 1).
While advancing our characterization of key players in the process of RNA-templated DSB repair in yeast cells, we developed new genetic systems to characterize DSB repair mechanisms by RNA human cells. Our results point towards an unexpected function of RNA in directing the way DSBs are repaired in human cells.
Features of ribonucleotides embedded in genomic DNA. RNA and DNA interact with each other in many fundamental cellular processes, such as DNA replication, transcription, and DNA repair, as we have shown for RNA-templated DSB repair. An even more intimate connection between the two nucleic acids is that DNA can contain the units of RNA, ribonucleoside monophosphates, rNMPs. It has been known for some time that rNMPs are present at specific loci in mouse and human mitochondrial DNA (Grossman et al., 1973), at the mating type locus of fission yeast (Vengrova and Dalgaard, 2006), and even in chloroplast DNA (Kolodner et al., 1975). However, it is only in the last decade that the presence of rNMPs has been revealed to be a general feature of DNA in nature (Caldecott, 2014; Cavanaugh et al., 2010; Nick McElhinny et al., 2010; Williams and Kunkel, 2014; Williams et al., 2016). Over one million rNMPs are incorporated in the mouse genome every cell cycle (Reijns et al., 2012), and several thousand rNMPs in the budding yeast genome (Williams and Kunkel, 2014). Nearly all DNA polymerases incorporate rNMPs into DNA under physiological conditions (Astatke et al., 1998; Bonnin et al., 1999; Brown and Suo, 2011; Cavanaugh et al., 2011; Cavanaugh et al., 2010; Gong et al., 2005; Kasiviswanathan and Copeland, 2011; McDonald et al., 2012; Nick McElhinny and Ramsden, 2003; Patel and Loeb, 2000). Moreover, the levels of rNMPs found in DNA are striking with a frequency of 1 in 700 (Nava et al., 2020; Sparks et al., 2012). Despite the strong sugar discrimination capacity of replicative DNA polymerases (DeLucia et al., 2003; Joyce, 1997), rNMPs outnumber common depurination and base oxidation sites together by more than an order of magnitude (Wallace and Williams, 2014) and are thus the most abundant non-canonical nucleotides found in cellular DNA (Caldecott, 2014; Nava et al., 2020; Williams and Kunkel, 2014).
The 2′-hydroxyl group in the ribose sugar of rNMPs in DNA alters DNA properties and structure (Ban et al., 1994; Chiu et al., 2014; DeRose et al., 2012; Egli et al., 1993; Jaishree et al., 1993; Koh et al., 2015b), and increases DNA fragility and mutability (Caldecott, 2014; Klein, 2017; Potenski and Klein, 2014; Wallace and Williams, 2014). In nuclear DNA, rNMPs are often removed soon after they are incorporated during DNA replication. The enzyme ribonuclease (RNase) H2 initiates ribonucleotide excision repair (RER), which is the major cellular mechanism that removes rNMPs embedded in nuclear DNA (Sparks et al., 2012). Paired and mis-paired rNMPs in DNA can also be targeted by nucleotide excision repair in bacteria (Vaisman et al., 2013), and mismatch repair (Shen et al., 2011), respectively.
Most of our knowledge about rNMPs in DNA links rNMPs to genome instability and fragility and derives from studies in which RNase H2 activity is inhibited, wherein the negative consequences of the unremoved rNMPs are exacerbated (Cerritelli and El Hage, 2020; Cerritelli et al., 2020; Klein, 2017; Takeishi et al., 2020; Zong et al., 2020). Null RNase H2 is embryonic lethal (Cerritelli and Crouch, 2009). In the presence of mutated RNase H2 genes, DNA-embedded rNMPs cause DNA damage and increased sensitivity to cancer inhibitors (Zimmermann et al., 2018). RNase H2-null murine embryonic fibroblasts accumulate over 1 million rNMPs in their genomic DNA, activating a DNA damage response (Reijns et al., 2012). In humans, mutations in three subunits of RNase H2 are associated with the neurological Aicardi-Goutieres syndrome (AGS), which causes severe brain dysfunction; most patients do not survive past childhood (Crow et al., 2006; Rice et al., 2007). The defective removal of rNMPs from DNA by mutants of RNase H2 of AGS patients may activate an autoimmune response (Gunther et al., 2015). Moreover, recent studies have linked rNMP incorporation to other pathologies, such as cancer, in mouse models (Aden et al., 2019; Hiller et al., 2018; Moss et al., 2017).
Six years ago, the development of high-throughput sequencing approaches to map the precise position of rNMPs in the genomes of cells marked a major step towards understanding function and consequences of rNMPs in DNA. My group in parallel with three other groups developed genomic tools to map rNMPs in DNA (Clausen et al., 2015; Daigaku et al., 2015; Jinks-Robertson and Klein, 2015; Koh et al., 2015a; Reijns et al., 2015). We are now poised to obtain significant knowledge about rNMPs and their relationship with the genome. Very little is known about rNMPs in human chromosomal DNA.
We developed two unique tools that are key to study the significance of rNMPs in DNA: ribose-seq (experimental protocol) and Ribose-Map (bioinformatics software). The ribose-seq technique enables construction of libraries of rNMP sites that are present in any DNA of interest (Koh et al., 2015a). The ribose-seq protocol was recently advanced and optimized (Balachander et al., 2019) (Figure 2). Briefly, after genomic DNA extraction, the DNA is cut using blunt-end restriction enzymes (REs). A dA-tailing reaction is performed to allow ligation of specific adaptors containing a unique molecular identifier of 8 random nucleotides to the blunt-ended genomic fragments. The fragments are then treated with alkali, which denatures the DNA and nicks 3′ to each rNMP in DNA. Ribose-seq captures DNA fragments with terminal 2′,3′-cyclic phosphates (2′,3′-cP) or 2′-phosphates (2′-P) generated by alkaline cleavage at rNMP sites via the self-ligation of single stranded DNA products by the Arabidopsis thaliana tRNA ligase (AtRNL) (Koh et al., 2015a). Following exonuclease treatment to degrade the background of unligated products, and treatment with 2′-phosphotransferase Tpt1 to remove the 2′-P remaining at the ligation junction, circular molecules are PCR-amplified in 2 rounds to generate a library of linear DNA molecules, each containing the site of rNMP incorporation and its upstream sequence (PCR 1), and to attach indexes for Illumina sequencing (PCR 2). The library is then size selected to eliminate primer dimers, and purified before sequencing.
Ribose-Map is a standardized bioinformatics toolkit that can map the genome-wide distribution of rNMPs embedded in DNA independently of the rNMP sequencing technique used (Gombolay et al., 2019). Ribose-Map transforms raw rNMP sequencing data into summary datasets and publication-ready visualizations of results. Ribose-Map has been outfitted with six analytical modules for preparing the reads for analysis, locating the genomic coordinates of rNMPs, exploring the genome-wide distribution of rNMPs, determining the nucleotide sequence context (with various length) of rNMPs, and identifying hotspots of rNMP incorporation (Gombolay and Storici, 2020). Ribose-Map is available as github depository at https://github.com/agombolay/ribose-map.
Recently, we built and analyzed over 50 ribose-seq libraries with mitochondrial and nuclear DNA derived from three yeast species (Saccharomyces cerevisiae, Saccharomyces paradoxus and Schizosaccharomyces pombe) using several different common lab strains of wild-type and mutant genotypes of RNase H2. In our yeast study, we found that genomic rNMP incorporation is not random; it is markedly driven by sequence context, specifically by the sequence of the deoxyribonucleoside monophosphate (dNMP) immediately upstream of the site of incorporation. Our results suggest a mechanism of structural accommodation of the rNMPs in the active site of replicative DNA polymerases. We consistently found dAMP immediately upstream of the most abundant genomic rCMPs and rGMPs. dArC and dArG were dominant signatures in short-nucleotide repeats that we found to be hotspot sites of rNMP incorporation (Figure 3).
This study provides a model to uncover features and functions of genomic rNMP incorporation in all forms of life. We have also found non-random incorporation of rNMPs, as well as rNMP patterns in the ribose-seq libraries of the green alga Chlamydomonas reinhardtii (El-Sayed et al., 2021). rNMP mapping in the genome of C. reinhardtii represents the first characterization of rNMPs in the DNA of a non-opisthokont (photosynthetic eukaryote or protist) organism (El-Sayed et al., 2021). The discovery of rNMP patterns in the nuclear DNA of yeast and algal species has led us to hypothesize that the presence of (mis)incorporated rNMPs, which represent a threat to genome stability especially in large genomes like the human genome, may be compensated by signalling functions residing in latent rNMP motifs.
Recently, we developed a series of scripts that are restriction enzyme set and combination optimization tools for rNMP capture techniques (RESCOT) to identify restriction enzyme (RE) combinations that produce ~300-600 bp blunt-ended fragments covering 62% to 77% of the human nuclear genome (Xu and Storici, 2021). Our recent bioinformatics studies using a multiplicity of ribose-seq, as well as emRiboSeq and RHII-HydEn-seq libraries from yeast S. cerevisiae have led us to discover that while the composition of rNMPs (rA/rC/rG/rU) is strikingly similar on both DNA strands around yeast origins of replication (autonomous replicating sequences, ARSs), the patterns of rNMP incorporation distinctly change on the leading and lagging strands at different distances from the ARSs (Xu and Storici, 2020). The results correlate markedly with the division of labor in DNA synthesis by the different DNA polymerases that participate in yeast DNA replication (Xu and Storici, 2020). These findings reveal specific signatures of rNMPs introduced by different DNA polymerases in yeast DNA. It is not yet known whether similar signatures are present in human DNA.
Armed with the ribose-seq procedure to map rNMPs in DNA via next-generation sequencing and the Ribose-Map computational toolkit, we built and initiated analyses of rNMP libraries from a number of human cell types. Analyses are underway. For example, we found that the embedded rNMPs are widespread and specific patterns are found in human DNA. Our results are revealing major variation of rNMP characteristics in the mitochondrial DNA of the different eukaryotic genomes studied. On the contrary, we uncovered noticeable conservation of rNMP-incorporation features in the nuclear genomes of the cell types analyzed. Our research is posed to set the stage for the discovery of potential cryptic roles of rNMPs embedded in DNA, and for a better understanding of the biology of our genome.
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