Investigation of Transfer RNA-Modifying Enzyme Knockout Strains of S. cerevisiae

Abstract

Modified nucleotides in tRNA are diverse and abundant, and aberrations are implicated in several human diseases. However, the physiological role of these modified nucleotides remains poorly understood. Previous studies have shown that S. cerevisiae strains containing multiple knockouts of tRNA modifying enzymes display temperature sensitive growth due to degradation of hypomodified tRNAs through the rapid tRNA decay (RTD) pathway. Based on high throughput screens indicating negative genetic interactions, five single knockout and five double knockout strains were generated. Single knockout strains pus1-Δ, trm1-Δ, tan1-Δ, trm8-Δ, and maf1-Δ and double knockout strains pus1-Δ dus3-Δ and trm1-Δ dus2-Δ did not exhibit temperature sensitive growth. In contrast, double knockout strains, trm8-Δ dus3-Δ, tan1-Δ dus2-Δ, and maf1-Δ dus2-Δ exhibited temperature sensitive growth. tRNA sequencing was used to determine tRNA abundances to assess whether tRNA stability was affected in the tan1-Δ dus2-Δ strain. While sequencing results were largely inconclusive, this work provides a characterization of several knockout strains and a basis for further investigation into the tan1-Δ dus2-Δ strain.