Determination of the specificity of small molecule splicing modifiers acting on A-1 bulged 5’-splice sites and rational development of novel splicing modifiers.

Specific mRNA splicing correction using small molecules is an emerging field of drug discovery which has already provided innovative therapeutic applications in the context of inherited diseases. In the context of Spinal Muscular Atrophy and Hungtington’s disease, the small molecule acts as a molecular glue between the first particle of the spliceosome and an A_-1 bulged 5’-splice site to promote specific splicing correction and provide orally available treatments. Even if the mechanism of action of the first small molecule splicing modifiers was elucidated (Campagne S. et al., Nat. Chem. Biol. 2019; Malard F. et al., Nucleic Acids Research 2024) and the concept of 5’-splice site bulge repair was discovered, there are still some open questions to fully understand the gene selectivity of splicing modifiers targeting an A_-1 bulged 5’-splice site. Notably, in order to be sensitive to the small molecule, the exon has to contain an adenine in position -1 (as pointed by the 5’-splice site bulge repair mechanism) but has to contain as well a purine rich cis-RNA element in the exon, well known to recruit trans-splicing factors. In order to better understand the specificity of the A_-1 splicing modifiers, the PhD student will develop a protocol to reassemble the splicing correction complex (A_-1 splicing modifier, U1 snRNP, trans-splicing factors and a synthetic pre-mRNA fragment) on an in vitro transcribed mRNA fragment in commercial HeLa nuclear extract. The candidate will optimize the purification of the splicing correction complex in order to determine its composition using mass spectroscopy and its cryoEM structure. Altogether, we aim to shed the light on the quaternary structure stabilized by the A_-1 splicing modifier in order to fully uncover the gene selectivity of the synthetic splicing switch but also unprecedented insights into mRNA splicing regulation. In parallel, the PhD candidate will also participate to the group project aiming at diversifying the pool of small molecule splicing modifiers and will discover using state-of-art AI-powered methods novel splicing modifiers acting on U_-1 and C_-1 bulged 5’-splice sites. Since many diseases originate from mutations falling at the position -1 of the 5’-splice sites, the overall aim of the project is to further explore the fundamental basis of the small molecule splicing modifier gene specificity but also to rationally design new tools for biomedicine. This PhD project is funded by the FRM (Fédération de la Recherche Médicale) in the context of the Amorcage program and the expected starting date is the 1^st of September 2024.

The ARNA (ARN: Natural and Artificial Regulation, https://arna.cnrs.fr/) unit rassembles RNA biologists and chemists who study the molecular mechanisms enabling RNA metabolism regulation and develop synthetic effectors to manipulate RNA metabolism with therapeutic outcomes. The unit is composed of 150 researchers. Within the ARNA unit, the canditiate will integarte the PRISM (Protein and RNA interactions sudied by Structural Methods) team headed by Dr. Cameron Mackereth. The PRISM team is divided into 5 groups and among those, the candidate will join the SMaRT group (Structure Mechanism and RNA Therapeutics; https://www.rna-smart.com/). The SMaRT lab is located in the European Institute of Chemistry and Biology in Pessac (near Bordeaux) which is equipped by state of art structural biology facilities (NMR spectroscopy, cryo electron microscope, X-ray) and important equipment needed to perform high standard biochemical and biological studies (cell lab, biochemistry platform, ...). The thesis will be embbeded into the SVS doctoral school of the University of Bordeaux.  

We are seeking for an excellent student (having a Master or an high school engeneer diploma) who is willing to work at the interface between chemistry, biochemistry, drug discovery and structural biology in a young and dynamic group of research. Excellent skills in molecular biology, biochemistry and structural biology are expected. Previous experience in structure determination by cryoEM and a solid training in bioinformatics will be a nice add-on.