Poster abstracts
Poster number 59 submitted by Malithi Jayasinghe
Reverse (3'-5') RNA polymerases: Applications beyond tRNAHis maturation and non-coding RNA processing
Malithi I. Jayasinghe (Chemistry and Biochemistry, Ohio State University), Jane E. Jackman (Chemistry and Biochemistry, Ohio State University)
Abstract:
The recent discovery of the reverse (3'-5') RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), opened a whole new world of possibilities, with potential applications in developing novel nucleic acid 5'-end labeling tools that are not supported by traditional forward (5'-3') polymerases.1, 2 Previous studies on many TLPs from a variety of organisms have shown their flexibility in accommodating various RNA substrates, consistent with their varied physiological roles.3, 4 However, early studies on templated nucleotide addition catalyzed by this enzyme family focused mainly on nucleotide addition to highly structured RNAs such as tRNAs and non-coding RNAs. Therefore, a clear understanding of the nucleotide addition by the Thg1/TLP enzymes on other RNAs, especially on model unstructured RNA substrates was lacking. Previous work in our lab demonstrated the feasibility of using various members of the TLP family to incorporate specific 5'-nucleotides into a short (26 nucleotide) model RNA substrate in the presence of an RNA oligonucleotide that serves as a template for the nucleotide addition. The RNA oligonucleotide template in this initial system is complementary to the 5'-end of the model RNA substrate and creates a 3'-overhang with a nucleotide combination of interest that serves as the template for incorporation of specific nucleotides. As evidenced from early experiments using this system, some TLPs are capable of incorporating a given combination of nucleotides across from the tested templates. Thus, we hypothesize that Thg1/TLP enzymes are amenable to be engineered as a versatile nucleic acid (DNA or RNA) 5'-end labeling tool. However, general principles governing how well specific substrate/template sequences can be accommodated by different TLPs have not been defined. The goal of this project is to define these principles by determining optimal nucleotide preference that is required for efficient nucleotide incorporation by TLPs. Methods to overcome challenges associated with separation of resulting long, highly stable RNA duplexes for accurate analysis purposes will also be developed as a goal of this work.
References:
[1] Gu, W., Jackman, J. E., Lohan, A. J., Gray, M. W., and Phizicky, E. M. (2003) tRNA [His] maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNA [His], Genes & Development 17, 2889-2901
[2] Jackman, J. E., and Phizicky, E. M. (2006) tRNA (His) guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition, Proceedings of the National Academy of Sciences of the United States of America 103, 8640-8645
[3] Rao, B. S., Maris, E. L., and Jackman, J. E. (2011) tRNA 5'-end repair activities of tRNA (His) guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea, Nucleic Acids Research 39, 1833-1842
[4] Long, Y. C., Abad, M. G., Olson, E. D., Carrillo, E. Y., and Jackman, J. E. (2016) Identification of distinct biological functions for four 3'-5' RNA polymerases, Nucleic Acids Research 44, 8395-8406
Keywords: 3-5 polymerase, nucleic acid 5-end labeling, tRNAHis guanylyltransferase