Poster abstracts

Poster number 72 submitted by Madison Allegretti

Investigating molecular interactions with 3’-5’ RNA polymerases in Dictyostelium Discoideum

Madison Allegretti (The Ohio State University Chemistry and Biochemistry), Brandon Iwaniec (The Ohio State University Biochemistry Program), Jane E. Jackman (The Ohio State University Chemistry and Biochemistry)

The Thg1/TLP family of enzymes catalyze a number of diverse 3’-5’ nucleotide addition reactions that are involved in processes such as tRNA-His maturation and 5’ end repair of tRNAs. While Thg1 has a strong preference to add a G-1 nucleotide to the 5’ end of tRNA-His in a non-Watson-Crick pair, TLPs, or Thg1-like-proteins, kinetically prefer to repair truncated RNA substrates in a Watson-Crick dependent manner and are able to act upon a variety of tRNAs beyond tRNA-His. The eukaryotic slime mold Dictyostelium discoideum encodes four enzymes from the Thg1/TLP family: DdiThg1, DdiTLP2, DdiTLP3, and DdiTLP4. Previous work has revealed distinct biological functions for three out of the four DdiTLPs. Because of the complexity of tRNA editing and the diversity of the reactions catalyzed by the different TLP enzymes expressed in Dictyostelium, we hypothesize that these enzymes may interact with one or more cellular macromolecules in order to fulfill their respective biological functions. In this investigation we are focusing on DdiTLP3, which is targeted to the mitochondria of the cell where it repairs the 5’ end of mitochondrial tRNAs, and DdiTLP4, whose true in vivo function remains unclear but has demonstrated editing activity on a variety of RNA substrates. By encoding a FLAG epitope tag to a terminus of these Ddi TLPs, interacting proteins that participate in the reactions catalyzed by the TLPs in D. discoideum cells can be immunoprecipitated and identified. This approach will uncover unknown mechanistic details of the reactions that these 3’-5’ polymerases are involved in and will help us further distinguish the roles that each enzyme plays in the cell. By further understanding the interactions that occur between DdiTLP3 and DdiTLP4 and other candidate molecules, we can understand the exact roles these enzymes play in Dictyostelium and possibly apply our findings to the roles of TLPs in other eukaryotes where their function has not yet been demonstrated.

Abad MG, Long Y, Willcox A, Gott JM, Gray MW, Jackman JE. A role for tRNAHis guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5′-tRNA editing. RNA. 2011;17(4):613-623. doi:10.1261/rna.2517111

Jackman JE, Gott JM, Gray MW. Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily. RNA. 2012;18(5):886-899. doi:10.1261/rna.032300.112

Abad MG, Rao BS, Jackman JE. Template-dependent 3′–5′ nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life. Proc Natl Acad Sci U S A. 2010;107(2):674-679. doi:10.1073/pnas.0910961107

Long Y, Abad MG, Olson ED, Carrillo EY, Jackman JE. Identification of distinct biological functions for four 3′-5′ RNA polymerases. Nucleic Acids Research. 2016;44(17):8395-8406. doi:10.1093/nar/gkw681

Keywords: tRNA editing, 3-5 polymerase