Poster abstracts
Poster number 5 submitted by Thaanya Amarasekara
Substrate specificity and 3'-5'-polymerization capabilities of two tRNA His guanylyltransferase-like proteins
Thaanya Amarasekara (Department of Chemistry and Biochemistry, The Ohio State University), Malithi Ishara Jayasinghe (Department of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Department of Chemistry and Biochemistry,The Ohio State University)
Abstract:
The discovery of tRNAHis guanylyltransferase-like proteins (TLPs), which catalyze nucleotide addition in the opposite direction (3'-5') to all standard DNA/RNA polymerases, has raised the possibility of developing a versatile 3'-5'-polymerase for multiple biotechnology applications, such as RNA 5'-end labeling. Although the physiological substrates identified so far for TLPs have all been highly structured RNAs, such as tRNA, two TLPs isolated from Acanthamoeba castellanii (Aca) and Dictyostelium discoideum (Ddi), known as AcaTLP2 and DdiTLP4, catalyze efficient 3'-5'-template-dependent nucleotide addition to short RNA duplexes in vitro. As the first members of the TLP family to act on non-tRNA substrates, understanding the biochemical properties is essential for developing these enzymes as broadly active RNA labeling tools. Based on the flexibility in substrate specificity exhibited by these enzymes compared to the other TLPs studied so far, we hypothesize that AcaTLP2 and DdiTLP4 will exhibit preferences for specific sequences and structures rather than acting uniformly on all small duplex RNA substrates. To comprehensively characterize these biochemical preferences, we designed an RNA substrate pool with nine randomized nucleotides, generating more than 200,000 sequences that can form various RNA structures to assess 3'-5'-polymerization using a high-throughput in vitro 3'-5'-extension assay. AcaTLP2 and DdiTLP4 successfully catalyzed the time-dependent addition of variable numbers of nucleotides to RNAs in this substrate pool. Characterization of the extension products by isolation and RNA-seq is ongoing to identify the most efficient substrates. Further characterization of selected RNAs by filter binding and activity assays will reveal the biochemical preferences for 3'-5'-polymerization by AcaTLP2 and DdiTLP4.
References:
Jayasinghe, M. I.; Patel, K. J.; Jackman, J. E. Thg1 family 3′–5′ RNA polymerases as tools for targeted RNA synthesis. RNA 2024, 30 (10), 1315–1327.
Chen, A. W.; Jayasinghe, M. I.; Chung, C. Z.; Rao, B. S.; Kenana, R.; Heinemann, I. U.; Jackman, J. E. The role of 3′ to 5′ reverse RNA polymerization in tRNA fidelity and repair. Genes 2019, 10 (3), 250.
Keywords: RNA substrates, 3-5-polymerization, TLPs