Transfer RNAs (tRNAs) are subject to numerous posttranscriptional modifications including methylation, which can control tRNA folding, stability, and function in messenger RNA (mRNA) translation. Despite this growing awareness, the role of RNA modifications, the so-called ?epitranscriptome? in mediating brain functions remains largely unknown. N7-methylguanosine (m7G) is one of the most prevalent tRNA modifications. Recent studies have linked mutations in components of the in the METTL1-WDR4 m7G tRNA methyltransferase complex with a distinct form of microcephalic primordial dwarfism characterized by facial dysmorphism, brain malformation, and severe encephalopathy with seizures. WDR4, located at human chromosome 21q22.3 is also a candidate gene for some of the Down syndrome phenotypes including mental retardation caused by trisomy of this chromosomal region in human patients. Indeed, Wdr4 is one a handful of candidate genes whose overexpression in mouse influences learning and memory in a model of Down syndrome. Interestingly, monosomy of the same cluster of genes also influences cognition and hippocampal plasticity, supporting that the precise dosage of certain gene(s) within this chromosomal region is critical for normal brain function. The primary goal of this exploratory proposal is to develop methods to map m7G modification of tRNAs at single nucleotide resolution in mouse embryonic stem cells (mESCs), differentiated neurons, and in the adult mouse brain. The molecular and cellular role of METTL1-WDR4 in m7G modification, translation, and neuronal differentiation will also be investigated using a mouse embryonic stem cell model. Finally, the effects of altered WDR4 dosage on m7G tRNA modification, mRNA translation, and neuronal differentiation will be tested using Down syndrome patient-derived induced pluripotent stem cells (iPSCs). Successful completion of this work will provide essential tools and technologies to investigate the role of tRNA methylation in disorders of the brain.
The goal of this proposal is to develop new methods to characterize tRNA modifications in stem cells, neuronal cells, and brain tissue and will facilitate investigation of the role of tRNA methylation in mental illnesses.