Quantitative PCR measurement of tRNA 2-Methylthio modification for assessing type 2 diabetes risk

Abstract

BACKGROUND: Genetic variants in the human CDKAL1 (CDK5 regulatory subunit associated protein 1-like 1) gene have been associated with reduced insulin secretion and type 2 diabetes (T2D).CDKAL1is a methylthiotransferase that catalyzes 2-methylthio (ms2) modification of the adenine at position 37 (A37) of cytoplasmic tRNALys- (UUU). We investigated the ms 2-modification level of tRNALys(UUU) as a direct readout of CDKAL1 enzyme activity in human samples. METHOD: We developed a quantitative PCR (qPCR)-based method to measure ms2 modification. tRNA Lys(UUU) was reverse-transcribed with 2 unique primers: Reverse primer r1 was designed to anneal to the middle of this tRNA, including the nucleotide at A37, and reverse primer r2 was designed to anneal to the region downstream (3) of A37. Subsequent qPCR was performed to detect the corresponding transcribed cDNAs. RESULTS: The efficiency of reverse transcription of tRNALys(UUU) was ms2-modification dependent. The relative difference in threshold cycle number obtained with the r1 or r2 primer yielded the ms2-modification level in tRNALys(UUU) precisely as predicted by an original mathematical model. The method was capable of measuring ms2-modification levels in tRNALys(UUU) in total RNA isolated from human peripheral blood samples, revealing that the ms 2-modification rate in tRNALys(UUU) was decreased in individuals carrying the CDKAL1 genotype associated with T2D. In addition, the ms2-modification level was correlated with insulin secretion. CONCLUSIONS: The results point to the critical role of ms2 modification in T2D and to a potential clinical use of a simple and high-throughput method for assessing T2D risk. © 2013 American Association for Clinical Chemistry.