Poster abstracts
Poster number 41 submitted by Ashton Davey
Bioinformatics approach for the identification of a box C/D guide RNA responsible for the Nm39 modification of tRNA
Ashton J. Davey (Department of Chemistry & Biochemistry, Northern Kentucky University), Holly M. Funk (Department of Chemistry & Biochemistry, Northern Kentucky University), Michael P. Guy (Department of Chemistry & Biochemistry, Northern Kentucky University)
Abstract:
tRNA modifications play an important role in translation. The 2’-O-methylation of tRNA position 39 only occurs in multicellular eukaryotes, and the responsible enzyme is unknown. Prior studies in our lab have tested orphan methyltransferases but have not yet found the responsible enzyme. Alternatively, this modification could be carried out by a box C/D guide RNA, which is a class of small nucleolar RNA (snoRNA). We used bioinformatics scripts PLEXY and snoGlobe to evaluate potential targets. We have tested three tRNAs known to receive the Nm39 modification in humans against a database of known guide RNAs. Results show several potential guide RNAs which were then validated using an external dataset of naturally occurring RNA-RNA hybrids identified through high throughput methodologies CLASH and FLASH. We will test the requirement for guide RNAs in cultured cells by knocking genes down using RNA interference in yeast. We will then test for the presence or absence of 2’-O-methylation using partial base hydrolysis and primer extension. Additional experiments will utilize liquid chromatography-mass spectrometry (LC-MS) to detect the presence of 2’-O-methylation. MET22 will also be knocked down to preserve tRNA integrity. Future experiments will be conducted in human HAP1 cells. Only two guide RNAs have previously been shown to modify tRNA. This research seeks to illuminate potential non-canonical guide RNA-tRNA interactions as well as modifications of the tRNA anticodon loop.
References:
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3. Vitali P, Kiss T. Cooperative 2’-o-methylation of the wobble cytidine of human elongator tRNAmet(cat) by a nucleolar and a cajal bodyspecific box C/D RNP. Genes and Development. 2019;33(13–14):741–746. doi:10.1101/gad.326363.119
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Keywords: snoRNA, tRNA modifcation, bioinformatics