Poster abstracts

Poster number 28 submitted by Mahmud Hossain

Detection of Mitochondrial Transfer RNAs Import of Saccharomyces cerevisiae by their Signature Digestion Products

Mahmud Hossain (Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221), Cathy Cherenfant (Chemistry and Physics Dept., Florida Southern College, Lakeland, FL 33801), Patrick A. Limbach (Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221)

Abstract:
Mitochondria, though containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. Mitochondrial import of transfer ribonucleic acids (tRNAs) from the cytosol, if not universal, is widely spread among species, ranging from yeast to trypanosomatids and to plants. However, the identification of numerous specific tRNAs simultaneously in a complex mixture is challenging due to their similar physio-chemical properties. We present a ribonuclease-mediated cleavage coupled with MALDI-MS for the detection of cytosolic and mitochondrial tRNAs from Saccharomyces cerevisiae by their signature digestion products, which eventually pave the way to detect specific imported tRNAs in mitochondria.
A comparison of an organism’s complete complement of tRNA endonuclease digestion products yields a set of unique or “signature” digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. The mass spectrometric detection of any one or more of these signature products by multiple ribonucleases (RNase T1 and RNase A) from a complex mixture will confirm the presence of the cognate tRNA. We have already established and reported a method to detect specific prokaryotic tRNAs in a complex cellular ensemble by means of this signature digestion approach.
Transfer RNA detection was initially optimized using commercially available yeast tRNA mixtures. S. cerevisiae (YPH499) was grown in YEPD medium to an OD of 1.5-2.0. tRNA fractionation was optimized using hot phenol and SDS coupled with chloroform followed by digestion with ribonucleases. The digested mixtures were analyzed by MALDI-TOF MS and matched with their theoretical signature digestion products. Research is underway to separate and detect solely mitochondrial tRNAs from this organism. Once established, this methodology could be used to detect imported RNAs in other eukaryotes including human mitochondria.

Keywords: signature digestion products, tRNA import, Saccharomyces cerevisiae