Poster abstracts
Poster number 8 submitted by Madison Allegretti
Investigating molecular interactions with 3’-5’ RNA polymerases in Dictyostelium Discoideum discoideum
Madison Allegretti (Chemistry and Biochemistry, The Ohio State University), Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Chemistry and Biochemistry, The Ohio State University)
Abstract:
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 Ddi TLPs. Because of the complexity of tRNA editing and the diversity of the reactions catalyzed by the different TLP enzymes expressed in D. discoideum, 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 DdiTLPs, interacting RNA and proteins that participate in the reactions catalyzed by the TLPs in D. discoideum cells can bewill be immunoprecipitated in vitro and in vivo and subsequently 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 D. discoideum and possibly apply our findings to the roles of TLPs in other eukaryotes where their function has not yet been demonstrated.
References:
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
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
Wells, J., Farnham, P.J. Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods. 2002; 26(1): 48-56. doi: 10.1016/S1046-2023(02)00007-5.
Keywords: reverse polymerase, 3-5 polymerase, tRNA modification