Developing single nuclei tRNA-seq to profile tRNA diversity at the single cell level

Project Summary/Abstract Transfer RNA (tRNA) biogenesis is essential for determining the pool of tRNAs that set the translational state of cells, and defects in the tRNA biogenesis machinery are associated with several types of neurodegenerative diseases. While bulk tRNA-sequencing assays of tissues and cell cultures have revealed that tRNA expression

and modification are regulated in tissue and disease-specific manners, technology to date has hindered our ability to define tRNA processing, expression, or modification changes in neurodevelopment and neurodegeneration at the level of single cells, limiting important insight and therapeutic development. In this

application, we propose to develop and apply a novel tRNA sequencing method to stem cell derived neurons and cerebral organoids to reveal complex tRNA processing and modification patterns at the single cell level for the first time. We will utilize preexisting laboratory models to establish and optimize our

technology (Aim 1) before profiling cerebral organoids with pathogenic variants in CLP1 and isogenic controls (Aim 2). These findings will reveal the complexity of tRNA processing and modification at unprecedented cellular level resolution. In addition to neurodevelopment and neurodegeneration, our research will have broad impacts

on the study of tRNAs across all tissues, as it will be applicable to any frozen or fresh tissue source. Outside of neurobiology, it will also benefit other fields such as cancer biology and virology where tRNA modulation is known to be important.