I am a graduate student in Dr. Ashleigh Schaffer’s lab. Broadly, the lab seeks to understand molecular mechanisms of physiological and pathological neurodevelopment. In pursuing this goal, Dr. Schaffer and others have identified a pattern in which mutations in genes encoding RNA processing proteins often cause neurological disease. In one example, mutations in tRNA splicing endonucleases (TSEN), required for maturation of a subset of tRNAs, lead to intellectual disability. Since TSEN proteins are expressed in all cell types, an unanswered question is why disruption of this complex leads to a distinctly neurological phenotype. One hypothesis is that tRNA processing and expression varies between cell types, and susceptibility to TSEN mutations is dependent upon the specific repertoire of tRNAs utilized by the cell. Currently, a nuanced understanding of cellular variation in tRNA biology is precluded by a lack of methods that quantify tRNA expression at single cell resolution.
The first aim of my project is to develop a protocol for single-cell tRNA sequencing (sc-tRNA-seq). By enzymatically polyadenylating all RNA in the cell, I plan to capture and integrate polyadenylated tRNAs into established protocols optimized for single-cell mRNA sequencing. After validating the novel methodology with bioinformatic and wet lab approaches, my second aim will apply sc-tRNA-seq to investigate tRNA expression during neurodevelopment. I will knock down each of the four TSEN proteins in isogenic populations of human embryonic stem cells and differentiate these populations into cortical organoids comprised of a variety of progenitor and mature neuronal cell types. With this physiologically relevant model system, I plan to quantify, for the first time, similarities and differences in tRNA expression between diverse cell types in both normal and disease-states of human neurodevelopment.
Figure 1 (left): Cortical organoid at 3 weeks of development immunostained for markers of neural progenitors (PAX6, red) and mature neurons (MAP2, green). Nuclei were stained with DAPI (blue). Scale bar = 100 micrometers