Presentation Title

Bi-allelic mutations in aminoacyl-tRNA synthetase interacting multifunctional protein 2 are linked to severe developmental encephalopathy and cause attenuation of protein synthesis and impaired cell cycle progression

Abstract

Recessive mutations in both aminoacyl-tRNA synthetases (ARS) and aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs) are associated with severe neurodevelopmental phenotypes in humans. The ARS are an essential class of protein synthesis enzymes that catalyze the attachment of amino acids to tRNA, while AIMPs (AIMP1-3) are scaffolding proteins that bind eight distinct ARS enzymes within a macromolecular complex in the cytoplasm known as the multisynthetase complex (MSC). While the precise function of the MSC is still actively being investigated, previous work indicates that this complex functions to optimize protein synthesis by keeping components of the translational machinery in close proximity. Through collaboration with clinical and geneticist colleagues we have identified and analyzed novel AIMP2 mutations that cause a severe neurodevelopmental disorder, or developmental encephalopathy, characterized by cerebral atrophy, epilepsy, and cognitive impairment. Because the phenotype resulting from mutations in AIMP1 and AIMP2 is similar to that caused by mutations in various ARS genes, we hypothesize that the pathogenicity of AIMP2 mutations is a consequence of impaired MSC function. In this work, we characterized AIMP2 mRNA and protein expression in patient fibroblasts carrying premature stop codon and splice-site mutations in AIMP2. We found that while cell viability and apoptosis appear to be unaffected in these cells, protein synthesis levels are decreased, and cell cycle progression is disrupted. Using plasmids encoding GFP tagged Y25STOP and Y35 STOP mutations, we show that co-transfection of anticodon engineered ‘suppressor tRNAs’ promotes stop codon readthrough. Additionally, electroporation of suppressor tRNA into patient cells partially rescues AIMP2 levels in the context of the homozygous Y35STOP mutation. Our findings demonstrate that premature stop codon, splice-site, frameshift, and point mutations in AIMP2 can cause developmental encephalopathy through a loss-of function-effect and investigate gene-based therapies to restore AIMP2 protein levels.

Primary Faculty Mentor Name

Christopher Francklyn

Graduate Student Mentors

Patrick Mullen

Faculty/Staff Collaborators

Dr. Alicia Ebert (Faculty Collaborator)

Status

Undergraduate

Student College

College of Agriculture and Life Sciences

Program/Major

Biological Science

Primary Research Category

Biological Sciences

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Bi-allelic mutations in aminoacyl-tRNA synthetase interacting multifunctional protein 2 are linked to severe developmental encephalopathy and cause attenuation of protein synthesis and impaired cell cycle progression

Recessive mutations in both aminoacyl-tRNA synthetases (ARS) and aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs) are associated with severe neurodevelopmental phenotypes in humans. The ARS are an essential class of protein synthesis enzymes that catalyze the attachment of amino acids to tRNA, while AIMPs (AIMP1-3) are scaffolding proteins that bind eight distinct ARS enzymes within a macromolecular complex in the cytoplasm known as the multisynthetase complex (MSC). While the precise function of the MSC is still actively being investigated, previous work indicates that this complex functions to optimize protein synthesis by keeping components of the translational machinery in close proximity. Through collaboration with clinical and geneticist colleagues we have identified and analyzed novel AIMP2 mutations that cause a severe neurodevelopmental disorder, or developmental encephalopathy, characterized by cerebral atrophy, epilepsy, and cognitive impairment. Because the phenotype resulting from mutations in AIMP1 and AIMP2 is similar to that caused by mutations in various ARS genes, we hypothesize that the pathogenicity of AIMP2 mutations is a consequence of impaired MSC function. In this work, we characterized AIMP2 mRNA and protein expression in patient fibroblasts carrying premature stop codon and splice-site mutations in AIMP2. We found that while cell viability and apoptosis appear to be unaffected in these cells, protein synthesis levels are decreased, and cell cycle progression is disrupted. Using plasmids encoding GFP tagged Y25STOP and Y35 STOP mutations, we show that co-transfection of anticodon engineered ‘suppressor tRNAs’ promotes stop codon readthrough. Additionally, electroporation of suppressor tRNA into patient cells partially rescues AIMP2 levels in the context of the homozygous Y35STOP mutation. Our findings demonstrate that premature stop codon, splice-site, frameshift, and point mutations in AIMP2 can cause developmental encephalopathy through a loss-of function-effect and investigate gene-based therapies to restore AIMP2 protein levels.