Identification of potential RNA substrates for the 3’-5’ polymerase BtTLP with RNA-Seq

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The Ohio State University

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Reverse (3'-5') polymerases are a relatively new discovery and are found in all three domains of life. The in vivo function of many of these proteins remains ambiguous. BtTLP, a reverse polymerase from the soil bacterium, Bacillus thuringiensis, has recently come under investigation through structural, genetic, and biochemical analysis. The overall goal of this study is to develop a new form of RNA-Seq to identify potential substrates for BtTLP in an engineered system (a previously characterized strain of Saccharomyces cerevisiae (baker's yeast) expressing BtTLP), with the ultimate goal of elucidating a more complete understanding of these unusual 3'-5' polymerases in biology. This project is being accomplished both from the wet and computational aspects. To start, a library of all small RNAs (approximately less than 200 bp) from the engineered system has been generated and sequenced. A computational pipeline has been developed to process the large amount of data that comes from deep-sequencing experiments. It is expected that multiple new substrates will be identified based on previous in vitro biochemical studies and an observed growth defect in the engineered system when compared to an isogenic control. This approach has significant advantages over the laborious alternative of testing each RNA in the cell individually. Traditionally, RNA-Seq has been used to identify functional elements in the genome, but here it is being used to detect post-transcriptional 5' nucleotide addition.


Rustbelt RNA Meeting 2014 Poster Presenter Awardee


Bioinformatics Biochemistry RNA Biology