Dominant and Genome-Wide Formation of DNA:RNA Hybrid G-Quadruplexes in Living Yeast Cells

Abstract

AbstractGuanine-rich nucleic acids form G-quadruplex (G4) structures that play a critical role in cellular processes. Previous studies have mostly focused on monomeric intramolecular G4s with four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has never been confirmed in eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and G4-specific probing reveals that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s still form and even dominate at genomic G4 sites that are theoretically capable of forming the monomeric intramolecular DNA G4s by themselves. Compared to DNA G4s (dG4s), hG4s exhibit a wider range of kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing hG4s to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also provides a sensitive and currently the only method for detecting the structural form of G4s in living cells.SignificanceThe identification of hybrid G-quadruplexes (hG4s) has disclosed a previously unrecognized structural form of G4s as the most common and abundant G4 species in the yeast genome. It reveals not only a dominant rule governing the formation of G4s in eukaryotic genomes, but also a unique genotype that allows G4-mediated transcriptional regulation to take feedback from the output as input, thus allowing the creation of feedback loops at the transcriptome scale.