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Abstract

Gene expression is a multi-layered process, tightly regulated by several transcriptional and post-transcriptional mechanisms. While transcription is regulated for example by DNA methylations and chromatin modifications, the fate of RNA post-transcriptionally is modulated by RNA modifications on coding and non-coding RNAs. The accurate control of gene expression is essential to ensure proper cellular function and normal development. Its disruption can lead to diseases. The aberrant deposition of tRNA modifications has been closely linked to neuro-developmental disorders and mitochondrial-linked disorders. However, the precise underlying molecular mechanisms how tRNA modifications contribute to human neuro-developmental processes are still largely unknown. In my study, I focused on elucidating the influence of tRNA modifications in human early ectodermal differentiation which defines the origin of central nervous system development. I differentiated human embryonic stem cells into the major ectodermal cell types neuroectoderm, neural crest, cranial placode and non-neural ectoderm. To determine the landscape of tRNA modifications, including expression of tRNA modifying enzymes and tRNA anticodon pools, I performed mass spectrometry, RNA sequencing, tRNA pool sequencing and proteomics analysis. I observed, that while tRNA modifications and tRNA anticodon pools remained largely stable throughout differentiation, the tRNA modifying enzymes were strongly downregulated in ectodermal cells compared to pluripotent stem cells. This downregulation of tRNA modifiers was accompanied by reduced global and mitochondrial protein synthesis in differentiated cells. By performing a knockdown screen of 47 tRNA modifiers, I observed that mitochondrial tRNA modifying enzymes affected not only mitochondrial protein synthesis but also strongly affected global protein synthesis. The reduction in mitochondrial protein synthesis in ectodermal cells led to reduced mitochondrial metabolic and OXPHOS activity. Since protein synthesis requires high amounts of energy, reduction of mitochondrial activity and therefore, reduced energy production affected also global protein synthesis. In summary, I showed that human early ectodermal cell types are in a more quiescent state compared to pluripotent stem cells and downregulate tRNA modifying enzymes, protein synthesis and mitochondrial activity.