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

Abstract : RNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the selfassociation 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 af nity 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 con rmed (i.e., there is a signi cant level of false positive). This report aims to bene t from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Brie y, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identi ed 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 work ow uncovers new rG4 candidates whose rG4-folding was then con rmed in vitro using an array of established biophysical methods. Clearly, this work ow led to the identi cation of novel rG4s in a highly specic and reliable manner

Guanine nucleotide-binding protein-like 1 (GNL1) binds RNA G-quadruplex structures in genes associated with Parkinson’s disease

Abstract : 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 5ʹ untranslated regions (5ʹUTR) of mRNAs associated with Parkinson’s disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5ʹUTRs 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

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

International audienceRNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the selfassociation 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

Cross-Sectional Evaluation of Humoral Responses against SARS-CoV-2 Spike

International audienceSARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediate viral entry and are the main targets for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic, and public health interventions. Here, we perform a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. Most infected individuals elicit anti-Spike antibodies within 2 weeks of the onset of symptoms. The levels of receptor binding domain (RBD)-specific immunoglobulin G (IgG) persist over time, and the levels of anti-RBD IgM decrease after symptom resolution. Although most individuals develop neutralizing antibodies within 2 weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection