Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY/ABSTRACT Multiple cases of MEHMO syndrome, an X-linked intellectual disability syndrome caused by mutations in EIF2S3 that encodes a core member of the cellular protein synthesis machinery, have recently been identified. However, the causal links between mis-regulated protein synthesis and human disease remain poorly understood. EIF2S3 encodes the γ subunit of translation initiation factor 2 (eIF2γ) that, along with the eIF2 α and β subunits, GTP, and the initiator Met-tRNAiMet, plays a critical role in selection of the translation start site. Thus, patients with MEHMO syndrome are likely to experience changes in protein synthesis on both a global and gene specific level. The acronym MEHMO denotes the symptoms of this syndrome: Mental deficiency, Epilepsy, Hypogenitalism, Microcephaly, and Obesity. How changes in gene expression incurred through mutations in eIF2γ result in the presentation of MEHMO syndrome remains unknown. Thus, there is a critical need to determine the underlying molecular mechanism governing the phenotype and symptoms of MEHMO patients. The long-term goal of these studies is to identify key factors and gene expression changes that underlie MEHMO syndrome in order to develop improved strategies for treating patients. The overall objective in this application is to determine how mis-regulation of protein synthesis contributes to the symptoms of MEHMO syndrome. In Aim 1 ribosome profiling, an informatics-based, genome- wide approach, and additional validation techniques will be used to identify mRNAs subject to changes in start codon selection and translational efficiency in MEHMO-patient derived iPSCs expressing mutant eIF2γ or isogenic iPSCs expressing wild-type eIF2g. In Aim 2 wild-type and eIF2γ mutant iPSCs will be differentiated into neurons that will be utilized in multidisciplinary analyses, including electrophysiological readings, immunohistochemistry, and ribosome profiling, geared toward understanding changes in neuronal biology and function caused by the eIF2γ mutation. In Aim 3 CRISPR Cas9 genome editing will be used to generate isogenic iPSCs with Vanishing White Matter disease-causing eIF2B mutations. Generation of eIF2B mutant iPSCs from the isogenic wild-type control iPSCs utilized in Aim 1 will enable direct comparisons of the protein synthesis changes caused by mutations in eIF2γ and its guanine-nucleotide exchange factor eIF2B. These studies will enhance our understanding of how translation is mis- regulated by alterations in eIF2 function, and ultimately provide critical knowledge for the development of targeted strategies for the treatment of patients with MEHMO syndrome.
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