De novo missense mutations in E3 ubiquitin ligase RNFT2 lead to intellectual disability as evidenced by loss of function studies in Drosophila
Authors: Ayşe Kahraman 1, 2; Barış Can Mandacı 1, 2; Reza Ataei 1, 3; Kenneth Schöneck 1; Anastasia Fokina 1, 2; Çiğdem Soysal 1; Elif Darbuka 1, 2; Ibrahim Yaman 1, 2; Kimia Kahrizi 4; Hossein Najmabadi 4; Mohammad Haddadi 3; Arzu Çelik 1, 2
Affiliations: 1) Bogazici University, Department of Molecular Biology and Genetics, Bebek, Istanbul, Turkey; 2) Bogaziçi University, Life Sciences Center, Bebek, Istanbul, Turkey; 3) University of Zabol, Department of Biology, Faculty of Science, Zabol, Iran; 4) University of Social Welfare and Rehabilitation Sciences, Genetics Research Center, Tehran, Iran
Keywords: a. neural degeneration; f. learning/memory
Characterized by various deficiencies in both intellectual ability and adaptive behavior, intellectual disabilities (IDs) affect 1% of the population worldwide. Mutations in over 400 genes have been implicated in ID, however the underlying molecular links between genotype and phenotype for most of these genes remain unknown. Whole exome sequencing (WES) of a large cohort of 404 Iranian consanguineous families with autosomal recessive intellectual disability (ARID) led to the identification of a novel likely pathogenic missense variant (cT1150C; p.C384R) in the RNFT2 gene. RNFT2 (RING finger transmembrane domain containing protein 2, also named as TMEM118), encodes a RING finger E3 ubiquitin ligase. Its fly ortholog dmel/CG13605 has the same type of RING domain (C3HC4 type) and is predicted to be a ubiquitin E3 ligase involved in the ER-regulated protein degradation pathway.
How disruption of RNFT2 gives rise to ID phenotypes is not known. To address this question, we sought to investigate the neurobehavioral consequences of losing the RNFT2 homolog in flies and identify its expression in the fly brain. In parallel, we investigated its subcellular localization in stably transfected HeLa cells. These cells will be used to identify the substrates of RNFT2.
Towards this end we have generated a Gal4 line and show that CG13605 is expressed in a subset of Kenyon cells in the fly brain starting at larval stages. Downregulation of CG13605 in the mushroom body with OK107-Gal4 lead to neurodevelopmental phenotypes such as loss of alpha and alpha’ lobes. For a more detailed functional characterization, we generated null mutant flies using the CRISPR/Cas9 system. Loss of CG13605 led to phenotypes such as alpha lobe loss or beta lobe fusion. The observed defects in mushroom body development, which is the primary memory and learning center of flies, provide evidence for RNFT2 being an ID gene. Ongoing experiments including rescue experiments, analyses using patient-specific variants of RNFT2 as well as behavioral studies are expected to further support this hypothesis.