Detecting chemical modifications of Ribonucleic Acid
AbstractTransfer Ribonucleic acid (tRNA) is one of the highly chemically modified RNA in the cell, and the isomerization of uridine to pseudouridine (ᴪ) is the most abundant RNA modification found in all domains of life. Although the function of RNA modification is not yet fully understood, the chemical changes in RNA have been implicated in cellular fitness and development of genetic diseases. For example, the bacterial proteins TruA and RluA are both pseudouridine synthases which catalyze the isomerization of uridine to pseudouridine. TruA is capable of modifying several nearby sites in tRNAs while RluA modifies different RNAs at positions with a shared sequence and structure. These modifications are conserved in all forms of life, but little is known about their function.
The objective of this research project was to modify tRNA using modification enzymes and to establish a method to detect multiple RNA modifications at once using High Performance Liquid Chromatography (HPLC). Using a reverse phase chromatography column will allow to separate samples based on hydrophobicity. On one side, tRNA was successfully in vitro transcribed, purified and digested into nucleosides, which were then separated using the HPLC column. On the other side, TruA and RluA wild-type proteins were successfully overexpressed and purified, in order to prepare modified tRNA. In the future, this system will enable us to detect and quantify the presence of pseudouridine as well as other chemical modifications in any type of RNA. This tool will thereby greatly advance research in the Kothe lab to understand the mechanisms and functions of RNA modifications.