Partho Sarothi RAY
SABNP Laboratory | Inserm U1204 | Université d’Evry-Paris-Saclay

Title: m6A, microRNA and RBP: The “Three Musketeers” of mRNA Regulation

Abstract: N6-methyladenosine (m6A), the most prevalent epitranscriptomic modification in eukaryotes, is enriched in 3′- untranslated regions (3′ UTRs) of mRNAs. m6A modifications directly determine the binding of a class of RNA-binding proteins (RBPs) called as m6A “readers” to RNA. However, as 3′ UTRs are major binding sites of RNA-binding protein (RBPs) and microRNAs (miRNAs), we hypothesized that m6A-dependent local RNA structure changes may further influence the proximal binding of RBPs and miRNAs and thereby regulate mRNA function. Using a human transcriptome-wide bioinformatic analysis, we found a strong positive correlation between number of m6A sites, miRNAs and RBPs binding to mRNAs, suggesting m6A-modified mRNAs are more targeted by miRNAs and RBPs. Moreover, m6A sites were also found to be located proximally to miRNA target sites and binding sites of multiple RBPs. This indicated three-way crosstalk between m6A, microRNA and RBP binding, suggesting the influence of mRNA modifications on the miRNA and RBP interactomes. From the subset of mRNAs which show proximity between m6A sites, target sites of the miRNA miR-125b and binding sites of the RBP HuR, we investigated COX7A2L, which encodes a crucial regulatory component of the mitochondrial respiratory chain and is upregulated in estrogen-treated breast cancer cells. We found that expression of COX7A2L is regulated by both miR-125b and HuR, and m6A modification facilitates the binding of miR-125b to the COX7A2L mRNA 3’UTR. However, HuR binds to the COX7A2L mRNA 3’UTR in absence of m6A modification and antagonizes the miR-125b mediated repression of COX7A2L expression. Hence, our observations have established that m6A modifications influence the interplay between miRNAs and RBPs in binding with mRNAs, with important consequences for the integrated post-transcriptional regulation of gene expression.

Maëlle Etienne (Bruno Sargueil’s lab)

Title: Positional mapping of lipid-RNA adduct formation in LNP and structural implications for vaccine mRNA

Authors:
Maëlle ETIENNE 1,2, Christelle KRAL 2, Nour AL-TURIHI 2, Isabelle LEGASTELOIS 2, Delphine ALLOUCHE 2, Bruno SARGUEIL 1
1 UMR 8038 CITCOM, 4 Avenue de l’observatoire, 75006 Paris
2 Sanofi mRNA Center of Excellence, 1541 Avenue Marcel Mérieux, 69210 Marcy L’Etoile, France

Abstract: Lipid nanoparticles (LNPs) encapsulate and protect mRNA through self-assembly of four lipid types, facilitating cellular internalization and cytoplasmic protein translation. Recent studies have revealed spontaneous formation of lipid-mRNA adducts within LNPs, potentially impacting mRNA structure and protein expression [1] . Notably, the proportion of these adducts increases over time. This research specifically investigates an mRNA encoding hemagglutinin subtype H3 (HAH3) from the Singapore strain. The experimental strategy combines two complementary approaches.

First, the native secondary structure of this mRNA was determined using SHAPE [2] (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension), providing the initial RNA secondary structure before LNP formulation. SHAPE allows investigating nucleotide flexibility through selective acylation of the 2'OH ribose groups in flexible regions. These SHAPE adducts block reverse transcription, generating cDNA fragments of various lengths that are then analyzed by capillary electrophoresis. Second, applying similar principles, a mapping of lipid-mRNA adduct formation sites was performed on the mRNA extracted from LNPs. After forcing mRNA-LNP adduct formation through increasing incubation times at 25°C, mRNA-LNPs are deformulated and analyzed using SHAPE workflows.

By correlating adduct positions with the native secondary structure of HAH3 mRNA, kinetic data reveal preferential mRNA-lipid adduct formation sites. These sites depend on both nucleotide type and structural position within the RNA molecule. This mapping provides insights into how lipid-mRNA interactions evolve over time and potentially affect RNA functionality within LNP delivery systems.

References : [1] Packer M, Gyawali D, Yerabolu R, Schariter J, White P. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat Commun. 2021 Nov 22;12(1):6777. doi: 10.1038/s41467-021-26926-0. PMID: 34811367; PMCID: PMC8608879. [2] Smola MJ, Rice GM, Busan S, Siegfried NA, Weeks KM. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc. 2015 Nov;10(11):1643-69. doi: 10.1038/nprot.2015.103. Epub 2015 Oct 1. PMID: 26426499; PMCID: PMC4900152.