Poster Title

Using RNA Interference for the Identification of the acp3U Enzyme in Higher Eukaryotes

Grade Level at Time of Presentation

Senior

Major

Chemistry

Minor

Biology

Institution

Northern Kentucky University

KY House District #

96

KY Senate District #

18

Department

Dept. of Biochemistry

Abstract

Cellular enzymes form dozens of post-transcriptional tRNA modifications to increase tRNA stability and function. tRNA modification defects are linked to various diseases, including intellectual disability and cancer. The enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) tRNA modification, which is found in animals and plants, but not in yeast, has not been identified. Because of its high conservation among plants and animals, acp3U is likely to be important for tRNA function. To identify the acp3U enzyme, we are silencing candidate genes in cultured Drosophila melanogaster cells by RNA interference (RNAi), analyzing acp3U levels on tRNA from treated cells using primer extension, and then verifying results using quantitative real time PCR (qRT-PCR). Candidate genes are identified through BLAST searches of predicted human methyltransferase genes of unknown function that are also found in D. melanogaster and plants, but not in yeast. To date, we have identified 30 candidate genes, treated cells with double stranded RNA (dsRNA) to induce RNAi for over half of these genes, and then analyzed tRNA from treated cells for the presence of acp3U by primer extension. To verify silencing of gene expression, qRT-PCR is being performed. Identification of the acp3U gene in animals, including humans, will increase our understanding of the link between tRNA modifications and disease.

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Using RNA Interference for the Identification of the acp3U Enzyme in Higher Eukaryotes

Cellular enzymes form dozens of post-transcriptional tRNA modifications to increase tRNA stability and function. tRNA modification defects are linked to various diseases, including intellectual disability and cancer. The enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) tRNA modification, which is found in animals and plants, but not in yeast, has not been identified. Because of its high conservation among plants and animals, acp3U is likely to be important for tRNA function. To identify the acp3U enzyme, we are silencing candidate genes in cultured Drosophila melanogaster cells by RNA interference (RNAi), analyzing acp3U levels on tRNA from treated cells using primer extension, and then verifying results using quantitative real time PCR (qRT-PCR). Candidate genes are identified through BLAST searches of predicted human methyltransferase genes of unknown function that are also found in D. melanogaster and plants, but not in yeast. To date, we have identified 30 candidate genes, treated cells with double stranded RNA (dsRNA) to induce RNAi for over half of these genes, and then analyzed tRNA from treated cells for the presence of acp3U by primer extension. To verify silencing of gene expression, qRT-PCR is being performed. Identification of the acp3U gene in animals, including humans, will increase our understanding of the link between tRNA modifications and disease.