Poster abstracts
Poster number 15 submitted by Samantha Dodbele
Characterization of Function and Mechanism of Recognition of an Orphan 3'-5' RNA Polymerase.
Samantha Dodbele (Ohio State Biochemistry Program), Yicheng Long (Ohio State Biochemistry Program), Jane Jackman (Ohio State Biochemistry Program, Center for RNA Biology)
Abstract:
Until the discovery of tRNAHis guanylyltransferase (Thg1) from Saccharomyces cerevisiae (Sc), nucleotide polymerization was believed to exclusively occur in the 5'-3' direction. Thg1 shifts this paradigm by catalyzing the non-templated addition of a required guanosine to the 5' end of tRNAHis in a 3'-5' direction. Enzymes exhibiting similarity to ScThg1, called Thg1-like proteins (TLPs) catalyze a Watson-Crick dependent 3'-5' polymerization. TLPs have been found in all three domains of life, including eukaryotic organisms such as Dictyostelium discoideum (Ddi). However, the roles and mechanisms of TLPs compared to their more well-studied Thg1 counterparts are less understood.
In D. discoideum, we have demonstrated that DdiThg1 and DdiTLP2 are responsible for cytosolic and mitochondrial tRNAHis maturation, respectively, and DdiTLP3 performs mitochondrial tRNA 5' editing, but the biological function of DdiTLP4 remains unknown. In vitro studies suggest DdiTLP4 can act on several small RNAs, including 5S ribosomal RNA, in addition to tRNAs. Moreover, depletion of DdiTLP4 causes a severe growth defect in D. discoideum. We hypothesize that this essential function of DdiTLP4 is due to its role in small RNA processing and its activity on specific non-coding RNA substrates unknown to us. Depletion of DdiTLP4 followed by RNA seq is being used to identify in vivo substrates of DdiTLP4, to provide the first insight into non-tRNA related activities associated with 3'-5' polymerases. In a related aim, we will use biochemical analysis of the activities of TLPs with substrates containing various 5' truncations to test the mechanism of RNA substrate recognition by these enzymes. Phosphatase protection assays will reveal whether the 3' overhang status is used as a ruler for recognition.
These investigations of biological function of DdiTLP4 and substrate selection by TLPs are significant, providing new insight into diverse biological roles and mechanisms of 3'-5' polymerization.
Keywords: Thg1, TLP, non-coding RNA