Poster abstracts
Poster number 8 submitted by Samantha Dodbele
Characterization of the Function of an Orphan 3'-5' RNA Polymerase Involved in Noncoding RNA Processing.
Samantha Dodbele (Ohio State Biochemistry Program, The Ohio State University OSU; Center for RNA Biology, OSU), Blythe Moreland (Center for RNA Biology, OSU; Department of Physics, OSU), Yicheng Long (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU), Ralf Bundschuh (Center for RNA Biology, OSU; Department of Physics, OSU; Division of Hematology and Department of Internal Medicine, OSU), Jane Jackman (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU; Department of Chemistry and Biochemistry, OSU)
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 relatively more well-studied Thg1 counterparts are less understood.
Previous work by our group has demonstrated the functions of two TLPs in the model eukaryote D. discoideum. These TLP enzymes, DdiTLP2, and DdiTLP3 catalyze a Watson-Crick dependent 3'-5' polymerization, and are responsible for mitochondrial tRNAHis maturation, and mitochondrial tRNA 5'-editing, respectively. However the biological function of a third TLP enzyme encoded in D. discoideum, DdiTLP4, remains unknown. In vitro studies suggest DdiTLP4 can act on several small, noncoding RNAs (ncRNA) in addition to tRNAs. Moreover, depletion of DdiTLP4 causes a severe growth defect in D. discoideum. Now we have the first evidence to suggest that the essential function of DdiTLP4 is due to its role in small RNA processing and its activity on specific ncRNA substrates. Depletion of DdiTLP4 followed by RNA-Seq was used to identify in vivo substrates of DdiTLP4 and enabling the identification of any type of RNA whose 5'-end sequence is altered in the absence of DdiTLP4 activity. This work comprises the first comprehensive insight into 3'-5' polymerase substrate specificity, including into non-tRNA related activities associated with these enzymes. Furthermore, investigation into the biological function of DdiTLP4 has provided greater understanding of the 5'-end maintenance machinery of eukaryotes and into diverse biological roles for 3'-5' polymerization.
Keywords: 3 to 5 polymerization, ncRNA, RNA-Seq