Poster abstracts
Poster number 50 submitted by Spencer Gardner
Identifying potential substrates of the reverse polymerase, BtTLP, by developing a variation on RNA-Seq
Spencer Gardner (Biomedical Engineering, The Ohio State University), Jane Jackman (Chemistry and Biochemistry, The Ohio State University), Ralf Bundschuh (Biophysics, The Ohio State University)
Abstract:
In this study, a variation on RNA-seq is being developed both from the bench and from the computer lab to discover potential small RNA substrates for the Thg1-like-protein (TLP) from Bacillus thuringiensis, called BtTLP. Thg1 and TLPs are a large family of reverse (3'-5') polymerases with various functions that revolve around tRNA repair and editing. Thg1 (tRNAHis guanylyltransferase) was discovered initially and its function is to add G-1 to tRNAHis in a non-Watson-Crick (W-C) manner, but TLPs prefer W-C polymerization, which may correspond to an ancestral activity found in the earlier members of this enzyme family. TLPs, being orthologs of Thg1, have been found in organisms from all three domains of life, including those that already contain a genomically encoded G-1 on tRNAHis. Surprisingly however, TLPs, including BtTLP, catalyze nucleotide addition in vitro on other 5' truncated RNAs that are not related to tRNAs in overall structure, such as 5S rRNA. This reaction begs the question of what the cellular substrates of 3'-5' reverse polymerases (BtTLP) are. The overall goal of this study is to investigate the roles of 3'-5' polymerases in biology.
This question is being investigated by isolating small RNA (<200 bp) from a Saccharomyces cerevisiae (baker's yeast) strain that has been engineered to express BtTLP. The resulting RNA is to be analyzed by high throughput deep sequencing, through a technique known as RNA-Seq. The massive amount of data (>106 reads per sample) that comes from this type of sequencing must be dealt with through a pipeline of various programs to process the data. The resultant processed genomic positions are analyzed on the UCSC Genome Browser to find peaks corresponding to different 5' locations on particular groups of reads. This approach is advantageous because it allows for essentially every RNA in the cell to be tested in a single experiment, rather than testing each RNA individually to determine whether it is a substrate for BtTLP-catalyzed nucleotide addition. This approach utilizes a novel way to use RNA-seq; which traditionally has been used to determine sequences that can be mapped back to the genome, here it is being used to discover post-transcriptional 5' nucleotide addition to small and perhaps 5'-truncated RNA molecules.
References:
1. Abad, Maria G., Bhalchandra S. Rao, and Jane E. Jackman. “Templated-dependent 3’-5’ nucleotide addition is a shared feature of tRNAHis guanylytransferase enzymes from multiple domains of life.” PNAS 107.2 (2010): 674-79. Print.
2. Jackman, Jane E., Jonatha M. Gott, and Michael W. Gray. “Doing it in reverse: 3’-to-5’ polymerization by the Thg1 superfamily.” RNA (2012): 886-99. Print.
3. Jackman, Jane E., and Eric M. Phizicky. “tRNAHis guanylyltransferase catalyzes a 3’-5’ polymerization reaction that is distinct from G-1 addition.” PNAS 103.23 (2006): 8640-45. Print.
4. Rao, Bhalchandra S., Emily L. Maris, and Jane E. Jackman. “tRNA 5’-end repair activates of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea.” Nucleic Acids Research (2010): 1-10. Print.
Keywords: RNA-Seq, Reverse Polymerase, Bioinformatics