tRNA Modifications and Development of Tools to Assess them
Central dogma in biology has expressed great emphasis on the order and function of various types of RNA and other biomolecules by assigning narrow processes based on one function. Transfer RNA (tRNA) is an example of such a narrow assignment, it is well known for one function: to carry amino acid isoforms to ribosomal active sites during translation. However, a variety of studies have shown that tRNA has a much larger and impactful role in gene regulation and the synthesis of proteins. tRNAs are highly modified RNAs. Regulation of modifications can occur during various forms of stress and diet, potentially leading to complex metabolic diseases as well as marked differences in various amino acid quantities. Furthermore, tRNAs have demonstrated the capacity to pass on those modifications to the progeny of the host organism through extracellular vesicles and sperm. The process of this is not well understood, as tRNA is technically challenging to sequence and study. Standard techniques do not capture the full sequence of the tRNA fragments. One reason for this is the presence of several methylated nucleotides which disrupt the process of reverse transcription. It is known that the obstacles that these methylation sites create can be bypassed by an Escherichia coli enzyme ALKB. This enzyme has shown promise as a useful tool to allow researchers to better understand full length and fragmented tRNA functionality. Our study focuses on the possibility that neurodegenerative diseases processes may have altered RNA modifications. Our first step to identifying changes in RNA modifications is to test our ability to detect the modifications using well-studied tRNAs as a model. We are using tRNAs from human brain with and without the ALKB treatment to assess our ability to detect regions of methylation and reverse transcription ‘stopping.’ Future implications for this tool development could be applied to many RNA biotypes across many diseases and injury states.