Canadian COVID-19 host genetics cohort replicates known severity associations.

The HostSeq initiative recruited 10,059 Canadians infected with SARS-CoV-2 between March 2020 and March 2023, obtained clinical information on their disease experience and whole genome sequenced (WGS) their DNA. We analyzed the WGS data for genetic contributors to severe COVID-19 (considering 3,499 hospitalized cases and 4,975 non-hospitalized after quality control). We investigated the evidence for replication of loci reported by the International Host Genetics Initiative (HGI); analyzed the X chromosome; conducted rare variant gene-based analysis and polygenic risk score testing. Population stratification was adjusted for using meta-analysis across ancestry groups. We replicated two loci identified by the HGI for COVID-19 severity: the LZTFL1/SLC6A20 locus on chromosome 3 and the FOXP4 locus on chromosome 6 (the latter with a variant significant at P < 5E-8). We found novel significant associations with MRAS and WDR89 in gene-based analyses, and constructed a polygenic risk score that explained 1.01% of the variance in severe COVID-19. This study provides independent evidence confirming the robustness of previously identified COVID-19 severity loci by the HGI and identifies novel genes for further investigation.

snoDB: an interactive database of human snoRNA sequences, abundance and interactions.

Small nucleolar RNAs (snoRNAs) are an abundant type of non-coding RNA with conserved functions in all known eukaryotes. Classified into two main families, the box C/D and H/ACA snoRNAs, they enact their most well characterized role of guiding site specific modifications in ribosomal RNA, through the formation of specific ribonucleoprotein complexes, with fundamental implications in ribosome biogenesis. However, it is becoming increasingly clear that the landscape of snoRNA cellular functionality is much broader than it once seemed with novel members, non-uniform expression patterns, new and diverse targets as well as several emerging non-canonical functions ranging from the modulation of alternative splicing to the regulation of chromatin architecture. In order to facilitate the further characterization of human snoRNAs in a holistic manner, we introduce an online interactive database tool: snoDB. Its purpose is to consolidate information on human snoRNAs from different sources such as sequence databases, target information, both canonical and non-canonical from the literature and from high-throughput RNA-RNA interaction datasets, as well as high-throughput sequencing data that can be visualized interactively.

Guanine Nucleotide-Binding Protein-Like 1 (GNL1) binds RNA G-quadruplex structures in genes associated with Parkinson's disease.

RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson's disease (PD) has not been analysed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5' untranslated regions (5'UTR) of mRNAs associated with Parkinson's disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5'UTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide-Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on new RNA G-quadruplexes located in genes dysregulated in Parkinson disease and a new G4-binding protein, GNL1.

Motif independent identification of potential RNA G-quadruplexes by G4RNA screener.

MOTIVATION: G-quadruplex structures in RNA molecules are known to have regulatory impacts in cells but are difficult to locate in the genome. The minimal requirements for G-quadruplex folding in RNA (G≥3N1-7 G≥3N1-7 G≥3N1-7 G≥3) is being challenged by observations made on specific examples in recent years. The definition of potential G-quadruplex sequences has major repercussions on the observation of the structure since it introduces a bias. The canonical motif only describes a sub-population of the reported G-quadruplexes. To address these issues, we propose an RNA G-quadruplex prediction strategy that does not rely on a motif definition. RESULTS: We trained an artificial neural network with sequences of experimentally validated G-quadruplexes from the G4RNA database encoded using an abstract definition of their sequence. This artificial neural network, G4NN, evaluates the similarity of a given sequence to known G-quadruplexes and reports it as a score. G4NN has a predictive power comparable to the reported G richness and G/C skewness evaluations that are the current state-of-the-art for the identification of potential RNA G-quadruplexes. We combined these approaches in the G4RNA screener, a program designed to manage and evaluate the sequences to identify potential G-quadruplexes. AVAILABILITY AND IMPLEMENTATION: G4RNA screener is available for download at http://gitlabscottgroup.med.usherbrooke.ca/J-Michel/g4rna_screener. CONTACT: jean-michel.garant@usherbrooke.ca or jean-pierre.perreault@usherbrooke.ca or michelle.scott@usherbrooke.ca. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

G-Quadruplexes influence pri-microRNA processing.

RNA G-Quadruplexes (G4) have been shown to possess many biological functions, including the regulation of microRNA (miRNA) biogenesis and function. However, their impact on pri-miRNA processing remains unknown. We identified G4 located near the Drosha cleavage site in three distinct pri-miRNAs: pri-mir200c, pri-mir451a, and pri-mir497. The folding of the potential G4 motifs was determined in solution. Subsequently, mutations disrupting G4 folding led to important changes in the mature miRNAs levels in cells. Moreover, using small antisense oligonucleotides binding to the pri-miRNA, it was possible to modulate, either positively or negatively, the mature miRNA levels. Together, these data demonstrate that G4 motifs could contribute to the regulation of pri-mRNA processing, a novel role for G4. Considering that bio-informatics screening indicates that between 9% and 50% of all pri-miRNAs contain a putative G4, these structures possess interesting potential as future therapeutic targets.

Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation.

G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.

The folding of 5'-UTR human G-quadruplexes possessing a long central loop.

G-quadruplexes are widespread four-stranded structures that are adopted by G-rich regions of both DNA and RNA and are involved in essential biological processes such as mRNA translation. They are formed by the stacking of two or more G-quartets that are linked together by three loops. Although the maximal loop length is usually fixed to 7 nt in most G-quadruplex-predicting software, it has already been demonstrated that artificial DNA G-quadruplexes containing two distal loops that are limited to 1 nt each and a central loop up to 30 nt long are likely to form in vitro. This report demonstrates that such structures possessing a long central loop are actually found in the 5'-UTRs of human mRNAs. Firstly, 1453 potential G-quadruplex-forming sequences (PG4s) were identified through a bioinformatic survey that searched for sequences respecting the requirement for two 1-nt long distal loops and a long central loop of 2-90 nt in length. Secondly, in vitro in-line probing experiments confirmed and characterized the folding of eight candidates possessing central loops of 10-70 nt long. Finally, the biological effect of several G-quadruplexes with a long central loop on mRNA expression was studied in cellulo using a luciferase gene reporter assay. Clearly, the actual definition of G-quadruplex-forming sequences is too conservative and must be expanded to include the long central loop. This greatly expands the number of expected PG4s in the transcriptome. Consideration of these new candidates might aid in elucidating the potentially important biological implications of the G-quadruplex structure.

Genome-wide detection of imprinted differentially methylated regions using nanopore sequencing.

Imprinting is a critical part of normal embryonic development in mammals, controlled by defined parent-of-origin (PofO) differentially methylated regions (DMRs) known as imprinting control regions. Direct nanopore sequencing of DNA provides a means to detect allelic methylation and to overcome the drawbacks of methylation array and short-read technologies. Here, we used publicly available nanopore sequencing data for 12 standard B-lymphocyte cell lines to acquire the genome-wide mapping of imprinted intervals in humans. Using the sequencing data, we were able to phase 95% of the human methylome and detect 94% of the previously well-characterized, imprinted DMRs. In addition, we found 42 novel imprinted DMRs (16 germline and 26 somatic), which were confirmed using whole-genome bisulfite sequencing (WGBS) data. Analysis of WGBS data in mouse (Mus musculus), rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) suggested that 17 of these imprinted DMRs are conserved. Some of the novel imprinted intervals are within or close to imprinted genes without a known DMR. We also detected subtle parental methylation bias, spanning several kilobases at seven known imprinted clusters. At these blocks, hypermethylation occurs at the gene body of expressed allele(s) with mutually exclusive H3K36me3 and H3K27me3 allelic histone marks. These results expand upon our current knowledge of imprinting and the potential of nanopore sequencing to identify imprinting regions using only parent-offspring trios, as opposed to the large multi-generational pedigrees that have previously been required.

Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.

Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.

Megabase-scale methylation phasing using nanopore long reads and NanoMethPhase.

The ability of nanopore sequencing to simultaneously detect modified nucleotides while producing long reads makes it ideal for detecting and phasing allele-specific methylation. However, there is currently no complete software for detecting SNPs, phasing haplotypes, and mapping methylation to these from nanopore sequence data. Here, we present NanoMethPhase, a software tool to phase 5-methylcytosine from nanopore sequencing. We also present SNVoter, which can post-process nanopore SNV calls to improve accuracy in low coverage regions. Together, these tools can accurately detect allele-specific methylation genome-wide using nanopore sequence data with low coverage of about ten-fold redundancy.

RNA G-Quadruplexes as Key Motifs of the Transcriptome.

G-Quadruplexes are non-canonical secondary structures that can be adopted under physiological conditions by guanine-rich DNA and RNA molecules. They have been reported to occur, and to perform multiple biological functions, in the genomes and transcriptomes of many species, including humans. This chapter focuses specifically on RNA G-quadruplexes and reviews the most recent discoveries in the field, as well as addresses the upcoming challenges researchers studying these structures face.

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.

G4RNA screener web server: User focused interface for RNA G-quadruplex prediction.

Though RNA G-quadruplexes became a focus of study over a decade ago, the main challenge associated with the identification of new potential G-quadruplexes remains a bottleneck step. It slows the study of these non-canonical structures in nucleic acids, and thus the understanding of their significance. The G4RNA screener is an accurate tool for the prediction of RNA G-quadruplexes but its deployment has brought to light an issue with its accessibility to G-quadruplex experts and biologists. G4RNA screener web server is a platform that provides a much needed interface to manage the input, parameters and result display of the main command-line ready tool. It is accessible at http://scottgroup.med.usherbrooke.ca/G4RNA_screener/.

Php4 Is a Key Player for Iron Economy in Meiotic and Sporulating Cells.

Meiosis is essential for sexually reproducing organisms, including the fission yeast Schizosaccharomyces pombe In meiosis, chromosomes replicate once in a diploid precursor cell (zygote), and then segregate twice to generate four haploid meiotic products, named spores in yeast. In S. pombe, Php4 is responsible for the transcriptional repression capability of the heteromeric CCAAT-binding factor to negatively regulate genes encoding iron-using proteins under low-iron conditions. Here, we show that the CCAAT-regulatory subunit Php4 is required for normal progression of meiosis under iron-limiting conditions. Cells lacking Php4 exhibit a meiotic arrest at metaphase I. Microscopic analyses of cells expressing functional GFP-Php4 show that it colocalizes with chromosomal material at every stage of meiosis under low concentrations of iron. In contrast, GFP-Php4 fluorescence signal is lost when cells undergo meiosis under iron-replete conditions. Global gene expression analysis of meiotic cells using DNA microarrays identified 137 genes that are regulated in an iron- and Php4-dependent manner. Among them, 18 genes are expressed exclusively during meiosis and constitute new putative Php4 target genes, which include hry1+ and mug14+ Further analysis validates that Php4 is required for maximal and timely repression of hry1+ and mug14+ genes. Using a chromatin immunoprecipitation approach, we show that Php4 specifically associates with hry1+ and mug14+ promoters in vivo Taken together, the results reveal that in iron-starved meiotic cells, Php4 is essential for completion of the meiotic program since it participates in global gene expression reprogramming to optimize the use of limited available iron.

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions.

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

G4RNA: an RNA G-quadruplex database.

G-quadruplexes (G4) are tetrahelical structures formed from planar arrangement of guanines in nucleic acids. A simple, regular motif was originally proposed to describe G4-forming sequences. More recently, however, formation of G4 was discovered to depend, at least in part, on the contextual backdrop of neighboring sequences. Prediction of G4 folding is thus becoming more challenging as G4 outlier structures, not described by the originally proposed motif, are increasingly reported. Recent observations thus call for a comprehensive tool, capable of consolidating the expanding information on tested G4s, in order to conduct systematic comparative analyses of G4-promoting sequences. The G4RNA Database we propose was designed to help meet the need for easily-retrievable data on known RNA G4s. A user-friendly, flexible query system allows for data retrieval on experimentally tested sequences, from many separate genes, to assess G4-folding potential. Query output sorts data according to sequence position, G4 likelihood, experimental outcomes and associated bibliographical references. G4RNA also provides an ideal foundation to collect and store additional sequence and experimental data, considering the growing interest G4s currently generate.

Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes.

G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.