Toward a Better Understanding of G4 Evolution in the 3 Living Kingdoms.

Background: G-quadruplexes (G4s) are secondary structures in DNA and RNA that impact various cellular processes, such as transcription, splicing, and translation. Due to their numerous functions, G4s are involved in many diseases, making their study important. Yet, G4s evolution remains largely unknown, due to their low sequence similarity and the poor quality of their sequence alignments across several species. To address this, we designed a strategy that avoids direct G4s alignment to study G4s evolution in the 3 species kingdoms. We also explored the coevolution between RBPs and G4s. Methods: We retrieved one-to-one orthologous genes from the Ensembl Compara database and computed groups of one-to-one orthologous genes. For each group, we aligned gene sequences and identified G4 families as groups of overlapping G4s in the alignment. We analyzed these G4 families using Count, a tool to infer feature evolution into a gene or a species tree. Additionally, we utilized these G4 families to predict G4s by homology. To establish a control dataset, we performed mono-, di- and tri-nucleotide shuffling. Results: Only a few conserved G4s occur among all living kingdoms. In eukaryotes, G4s exhibit slight conservation among vertebrates, and few are conserved between plants. In archaea and bacteria, at most, only 2 G4s are common. The G4 homology-based prediction increases the number of conserved G4s in common ancestors. The coevolution between RNA-binding proteins and G4s was investigated and revealed a modest impact of RNA-binding proteins evolution on G4 evolution. However, the details of this relationship remain unclear. Conclusion: Even if G4 evolution still eludes us, the present study provides key information to compute groups of homologous G4 and to reveal the evolution history of G4 families.

Development of a highly optimized procedure for the discovery of RNA G-quadruplexes by combining several strategies.

RNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the self-association of guanine quartets and that are stabilized by monovalent cations (e.g. potassium). rG4s are key elements in several post-transcriptional regulation mechanisms, including both messenger RNA (mRNA) and microRNA processing, mRNA transport and translation, to name but a few examples. Over the past few years, multiple high-throughput approaches have been developed in order to identify rG4s, including bioinformatic prediction, in vitro assays and affinity capture experiments coupled to RNA sequencing. Each individual approach had its limits, and thus yielded only a fraction of the potential rG4 that are further confirmed (i.e., there is a significant level of false positive). This report aims to benefit from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Briefly, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identified by RNA sequencing. Then, a novel bioinformatic pipeline that included DESeq2 to identify rG4 enriched transcripts, MACS2 to identify rG4 peaks, rG4-seq to increase rG4 formation probability and G4RNA Screener to detect putative rG4s was performed. This workflow uncovers new rG4 candidates whose rG4-folding was then confirmed in vitro using an array of established biophysical methods. Clearly, this workflow led to the identification of novel rG4s in a highly specific and reliable manner.

GAIA: G-quadruplexes in alive creature database.

G-quadruplexes (G4) are 3D structures that are found in both DNA and RNA. Interest in this structure has grown over the past few years due to both its implication in diverse biological mechanisms and its potential use as a therapeutic target, to name two examples. G4s in humans have been widely studied; however, the level of their study in other species remains relatively minimal. That said, progress in this field has resulted in the prediction of G4s structures in various species, ranging from bacteria to eukaryotes. These predictions were analysed in a previous study which revealed that G4s are present in all living kingdoms. To date, eleven different databases have grouped the various G4s depending on either their structures, on the proteins that might bind them, or on their location in the various genomes. However, none of these databases contains information on their location in the transcriptome of many of the implicated species. The GAIA database was designed so as to make this data available online in a user-friendly manner. Through its web interface, users can query GAIA to filter G4s, which, we hope, will help the research in this field. GAIA is available at: https://gaia.cobius.usherbrooke.ca.

G-quadruplex occurrence and conservation: more than just a question of guanine-cytosine content.

G-quadruplexes are motifs found in DNA and RNA that can fold into tertiary structures. Until now, they have been studied experimentally mainly in humans and a few other species. Recently, predictions have been made with bacterial and archaeal genomes. Nevertheless, a global comparison of predicted G4s (pG4s) across and within the three living kingdoms has not been addressed. In this study, we aimed to predict G4s in genes and transcripts of all kingdoms of living organisms and investigated the differences in their distributions. The relation of the predictions with GC content was studied. It appears that GC content is not the only parameter impacting G4 predictions and abundance. The distribution of pG4 densities varies depending on the class of transcripts and the group of species. Indeed, we have observed that, in coding transcripts, there are more predicted G4s than expected for eukaryotes but not for archaea and bacteria, while in noncoding transcripts, there are as many or fewer predicted G4s in all species groups. We even noticed that some species with the same GC content presented different pG4 profiles. For instance, Leishmania major and Chlamydomonas reinhardtii both have 60% of GC content, but the former has a pG4 density of 0.07 and the latter 1.16.

Where are G-quadruplexes located in the human transcriptome?

It has been demonstrated that RNA G-quadruplexes (G4) are structural motifs present in transcriptomes and play important regulatory roles in several post-transcriptional mechanisms. However, the full picture of RNA G4 locations and the extent of their implication remain elusive. Solely computational prediction analysis of the whole transcriptome may reveal all potential G4, since experimental identifications are always limited to specific conditions or specific cell lines. The present study reports the first in-depth computational prediction of potential G4 region across the complete human transcriptome. Although using a relatively stringent approach based on three prediction scores that accounts for the composition of G4 sequences, the composition of their neighboring sequences, and the various forms of G4, over 1.1 million of potential G4 (pG4) were predicted. The abundance of G4 was computationally confirmed in both 5' and 3'UTR as well as splicing junction of mRNA, appreciate for the first time in the long ncRNA, while almost absent of most of the small ncRNA families. The present results constitute an important step toward a full understanding of the roles of G4 in post-transcriptional mechanisms.