Investigating molecular interactions with 3'-5' RNA polymerases in Dictyostelium discoideum
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Date
2024-05
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The Ohio State University
Abstract
The Thg1/TLP family of enzymes catalyze a number of non-canonical 3'-5' nucleotide addition reactions across all domains of life. They play important roles in tRNA processing reactions such as tRNAHis maturation and 5' end repair of truncated tRNAs. Thg1, first discovered in yeast, has a strong preference to add a G-1 nucleotide to the 5' end of tRNAHis in a non-Watson- Crick base pair, permitting subsequent aminoacylation with histidine. TLPs, or Thg1-like-proteins, kinetically prefer to repair truncated RNA substrates in a Watson-Crick template dependent manner and demonstrate activity on many tRNA species beyond tRNAHis. This work focuses on two TLP enzymes found in the eukaryotic model organism Dictyostelium discoideum: DdiTLP3 and DdiTLP4. DdiTLP3 is targeted to the mitochondria of the cell where it repairs the 5' end of mitochondrial tRNA editing substrates as part of an incompletely understood mechanism. The true in vivo function of cytosolic DdiTLP4 remains unclear but it is an essential enzyme that has demonstrated polymerase activity on a variety of RNA substrates in vitro. Because of the complexity of tRNA editing and the diversity of the reactions catalyzed by Thg1/TLP enzymes encoded in D. discoideum, we hypothesize that DdiTLP3 and DdiTLP4 may interact with one or more cellular macromolecules in order to fulfill their respective biological functions. By identifying interacting molecules that facilitate their reactions, unknown mechanistic details of their 3'-5' nucleotide addition reactions can be elucidated. Encoding a FLAG epitope tag to a terminus of these DdiTLPs allows the capture of complexes of FLAG-tagged DdiTLP3 and DdiTLP4 with interacting RNA and proteins that participate in their reactions, which can then be identified with sequencing or mass spectrometry techniques. After concluding that the presence of a FLAG-tag does not impede on the activity of each TLP compared to wild-types, a protocol was optimized for in vitro crosslinking and immunoprecipitation (CLIP) with FLAG-tagged DdiTLP4 and total RNA isolated from D. discoideum. The efficiencies and specificities of two crosslinking methods, chemical and UV, were compared. In vivo CLIP with FLAG-tagged DdiTLP3 and DdiTLP4 in D. discoideum will further validate in vitro RNA target identification and also allow purification of interacting proteins. Together, these in vitro and in vivo approaches will enable identification of interacting RNAs and proteins that are important for the function of DdiTLP3 and DdiTLP4, thus providing insight into the mechanism and function of these unusual 3'-5' polymerases. 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.
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Keywords
tRNA modifications, RNA, 3'-5' polymerases