Investigating molecular mechanisms of RBMX-associated brain disorders and functional redundancy of RBMXL1 retrocopy in humans and mice

• Applicant: Professorin Christel Depienne, Ph.D.
• Subject Area: Developmental Neurobiology ; General Genetics and Functional Genome Biology ; Experimental Models for the Understanding of Nervous System Diseases ; Human Genetics ; Cognitive, Systems and Behavioural Neurobiology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 505514143

RBMX is a gene located on chromosome (chr) X in mammals that encodes the RNA-binding RMBX/hnRNP-G protein, known to regulate alternative splicing as part of the spliceosome machinery. Pathogenic variants in RBMX are associated with a severe but clinically variable neurodevelopmental disorder characterized by intellectual disability, brain and eye malformations affecting hemizygous males. Remarkably, RBMX has many copies in mammalian genomes, including several intronless retrocopies located on autosomes. The most recent retrocopies, RBMXL1 on human chr 1, and Rbmxl1a and Rbmxl1b on mouse chr 8 and 14, result from independent retroposition events and encode likely functional proteins, 96-98% identical to RBMX/Rbmx, expressed during brain development in both species. The main aims of this project are to 1) investigate the mechanisms by which novel human mutations identified in four unrelated families lead to brain disorders and 2) dissect the molecular mechanisms by which RBMX and its functional retrocopies contribute to brain development in both human and mouse. For this purpose, we will generate analogous, complementary cellular human and in vivo mouse models. On the one hand, we will generate iPSCs from fibroblasts of three patients and use CRISPR/cas9 genome editing to i) correct the mutation, and ii) inactivate RBMX, iii) RBMXL1 or iv) both. We will perform multiomics (RNAseq and proteomics) analysis in fibroblasts and neuronal precursors derived from iPSCs to identify genes and splicing isoforms controlled by RBMX and RBMXL1 and those altered by the mutations. We will also use minigene assays to test the ability of RBMX, RBMXL1 and RBMX mutant proteins to promote splicing in vitro and two-hybrid screens to identify protein partners either shared by or differing between the three human proteins. In parallel, we will use in utero electroporation of mouse embryos to investigate the cellular and molecular consequences of the knockdown of Rbmx and Rbmxl1 alone or in combination, and the ability of WT or mutant RBMX to rescue the observed defects. Our experimental strategy will allow testing non-exclusive hypotheses including loss- and gain-of-function of RBMX, as well as a dominant-negative impact on RBMXL1 as a result of genetic variants. A third part will consist in integrating results obtained in human and mouse models and disentangle species-specific contributions of RBMX/Rbmx and its retrocopies to brain developmental processes. In conclusion, this project is a pioneering undertaking that is very likely to provide groundbreaking insights into the complex mechanisms by which RBMX and its retrocopies control brain development. Furthermore, it has the potential to unravel how independent, recurrent retroposition of a single gene might have contributed to shape brain size and function during evolution.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Juliette Godin, Ph.D.