Biology subjects

Lejault, P.

Publications and source records attributed to Lejault, P..

5 recordsLinked to original sources

bioRxiv2024

Unraveling the Role of G-Quadruplexes in Alternative Polyadenylation (APA): A Focus on Neogenin 1

Polyadenylation is a crucial step in mRNA maturation. In half of the cases, alternative polyadenylation sites are used instead of canonical ones, affecting the length of the transcripts 3UTR. The mechanisms controlling the selection of these alternative polyadenylation sites are still unknown. Using a unique sequencing method (PolyAclick-seq) to identify different polyadenylated isoforms, we selected events modulated by RHPS4, a ligand known to stabilize G-quadruplex (G4). Through in silico selection, in vitro assays, and G4 mutagenesis construction, the pivotal role of rG4 structures in determining polyadenylation sites was uncovered, particularly for the gene encoding Neogenin-1 (NEO1). This research highlights the importance of focusing on G4 RNA mediated APA regulation in the 3UTR, as a method to alter isoform choice and impact protein synthesis opening up new avenues for RNA-based therapies.

biochemistry↗

bioRxiv2023

G-quadruplexe as a structural modulator of Intron Retention upon viral infection

Amongst the wide array of alternative splicing events (ASE), the intrinsic mechanisms of intron retention have remained elusive. This particular type of ASE has long been characterized as an artifact, but recent studies have shown its implication in numerous diseases. It has also been revealed that numerous viruses choose to disrupt alternative splicing to escape cellular immune response and further their proliferation. The main focus of this study was to investigate the G-quadruplex role in Alternative Splicing Events (ASEs) that occur following Flavivirus infections. After having demonstrated that G-quadruplexes structures are mainly formed in Intron Retained Transcripts by RNA-seq, our attention turned toward the ULK3 gene, coding for a serine/threonine kinase regulating autophagy, an essential mechanism in the cellular response to stress and even pathogen infections. In this study, we revealed the presence of a G-quadruplex in the first intron of the ULK3 gene near the 3 splice site. Furthermore, we assayed the formation and stability of this G-quadruplex in vitro and showed that its formation affects IR, as demonstrated by comparisons between wild-type and mutant transfected mini-genes. Finally, we identified the specific RNA-binding protein signature for this G-quadruplex, thereby uncovering the novel role of G-quadruplexes in Alternative Splicing.

biochemistry↗

bioRxiv2023

Small molecule chaperones facilitate the folding of long-non coding RNA

RNA G-quadruplexes (rG4) have recently emerged as major regulatory elements in both mRNA and non-coding RNA. To investigate the biological roles of the rG4 structures, chemists have developed a variety of highly specific and potent ligands. All these ligands bind to the rG4 by staking on their top, and the specificity of binding is demonstrated in comparison to other structures such as duplex or three-way junctions. It remains unclear whether rG4-ligands merely stabilize fully formed rG4 structures, or if they actively participate in the folding of the rG4 structure through their association with an unfolded RNA sequence. In order to access the innate steps of ligand-rG4 association and mechanisms, robust in vitro techniques, including FRET, electrophoretic mobility shift assay and reverse transcriptase stalling assays, were used to examine the capacity of five well-known G4 ligands to induce rG4 structures derived from either long non-coding RNAs of from synthetic RNAs. It was found that both PhenDC3 and PDS induce rG4 formation in unfolded single RNA strands. This discovery has important implications for the interpretation of RNA-seq experiments. Overall, in vitro data that can assist biochemists in selecting the optimal G4-ligands for their RNA cellular experiments are presented, while also considering the effects induced by these ligands of the rG4. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/535601v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@ec5191org.highwire.dtl.DTLVardef@df26ddorg.highwire.dtl.DTLVardef@707dc5org.highwire.dtl.DTLVardef@15117ea_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractA schematic representation of the potential structures that may arise from unfolded RNA that is rich in G nucleotides. rG4 can be stabilized by K+ either with or without G4 ligand and can be induced by a molecular chaperone in the presence of Li+.

biophysics↗

bioRxiv2020

Biomimetic, smart and multivalent ligands for G-quadruplex isolation and bioorthogonal imaging

G-quadruplexes (G4s) continue to gather wide attention in the field of chemical biology as their prevalence in the human genome and transcriptome strongly suggests that they may play key regulatory roles in cell biology. G4-specific, cell-permeable small molecules (G4-ligands) innovately permit the interrogation of cellular circuitries in order to assess to what extent G4s influence cell fate and functions. Here, we report on multivalent, biomimetic G4-ligands referred to as TASQs that enable both the isolation and visualization of G4s in human cells. Two biotinylated TASQs, BioTASQ and BioCyTASQ, are indeed efficient molecular tools to fish out G4s of mixtures of nucleic acids through simple affinity capture protocols and to image G4s in cells via a biotin/avidin pretargeted imaging system first applied here to G4s, found to be a reliable alternative to in situ click chemistry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/422878v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@2db02dorg.highwire.dtl.DTLVardef@1897e59org.highwire.dtl.DTLVardef@1c63716org.highwire.dtl.DTLVardef@4a8a5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Disclosing the actual efficiency of G-quadruplex DNA-disrupting small molecules

The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4s, the G4-unfold assay, and ii- fully characterize the first prototype of G4-disrupting small molecule, a phenylpyrrolcytosine (PhpC)-based G-clamp analog.

biophysics↗