Abstract 2091 Investigating molecular interactions with 3'-5' RNA polymerases in Dictyostelium Discoideum

The Thg1/TLP family of enzymes are 3'-5' RNA polymerases that catalyze diverse reactions during processes such as tRNAHis maturation and 5' end repair of tRNAs during tRNA editing. The eukaryotic slime mold Dictyostelium discoideum encodes four enzymes from the Thg1/TLP family: DdiThg1, DdiTLP2, DdiTLP3, and DdiTLP4. Previous work revealed distinct biological functions for three out of the four enzymes, while the biological role for DdiTLP4 remains unknown. Moreover, although it is known that DdiTLP3 catalyzes tRNA repair reactions during tRNA editing, the identity of the nuclease that generates tRNA substrates for tRNA repair in D. discoideum has not been identified. Therefore, the specific characteristics of tRNAs that are substrates for DdiTLP3 in vivo have also not been defined. We hypothesize that each of these enzymes interacts selectively with one or more cellular RNAs in order to carry out their respective biological functions. In this investigation, we are seeking to identify these RNAs that associate selectively with DdiTLP3 and DdiTLP4 to help define the biological substrates for each enzyme and to uncover unknown mechanistic details of their 3′-5′ nucleotide addition reactions. To do this, we carried out an immunoprecipitation-based approach to identify D. discoideum RNAs that co-purify with DdiTLP3 and DdiTLP4 that can be identified by RNA sequencing. I created constructs with a FLAG epitope tag fused to each terminus of both DdiTLPs for expression and purification in E. coli, and used kinetic assays to demonstrate that the presence of a FLAG-tag does not negatively impact the activity of each TLP compared to each wild-type enzyme. Subsequently, two different cross-linking methods, formaldehyde and UV light, were tested for the efficiency and specificity of protein-RNA crosslinks between the FLAG-tagged proteins and RNA isolated from D. discoideum. After optimization of each protocol, we determined that UV cross-linking, while less efficient, appears to result in a higher specificity of RNA-protein crosslinking. Utilizing radioactive 5'-end labeling, we have observed immunoprecipitated RNA of various apparent sizes under our optimized in vitro UV-CLIP conditions and will use RNA isolated from the CLIP reactions to identify these potential RNA substrates with RNA-Seq. Complementary experiments to perform CLIP to isolate co-purifying substrates with FLAG-tagged enzymes expressed in vivo in D. discoideum are also underway. By understanding the exact roles of TLPs in D. discoideum, we can possibly apply our findings to the roles of TLPs in other eukaryotes where specific biological functions for these enzymes have not yet been demonstrated.