Department of Molecular Physiology, Kumamoto University, Kumamoto, Japan;
Clin Chem. 2013 Nov;59(11):1604-12. doi: 10.1373/clinchem.2013.210401. Epub 2013 Aug 23.
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). CDKAL1 is a methylthiotransferase that catalyzes 2-methylthio (ms(2)) modification of the adenine at position 37 (A37) of cytoplasmic tRNA(Lys)(UUU). We investigated the ms(2)-modification level of tRNA(Lys)(UUU) as a direct readout of CDKAL1 enzyme activity in human samples.
We developed a quantitative PCR (qPCR)-based method to measure ms(2) 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.
The efficiency of reverse transcription of tRNA(Lys)(UUU) was ms(2)-modification dependent. The relative difference in threshold cycle number obtained with the r1 or r2 primer yielded the ms(2)-modification level in tRNA(Lys)(UUU) precisely as predicted by an original mathematical model. The method was capable of measuring ms(2)-modification levels in tRNA(Lys)(UUU) in total RNA isolated from human peripheral blood samples, revealing that the ms(2)-modification rate in tRNA(Lys)(UUU) was decreased in individuals carrying the CDKAL1 genotype associated with T2D. In addition, the ms(2)-modification level was correlated with insulin secretion.
The results point to the critical role of ms(2) modification in T2D and to a potential clinical use of a simple and high-throughput method for assessing T2D risk.
人类 CDKAL1（CDK5 调节亚单位相关蛋白 1 样 1）基因中的遗传变异与胰岛素分泌减少和 2 型糖尿病（T2D）有关。CDKAL1 是一种甲基硫转移酶，可催化细胞质 tRNA（Lys）（UUU）中腺嘌呤位置 37（A37）的 2-甲基硫（ms（2））修饰。我们研究了 tRNA（Lys）（UUU）的 ms（2）修饰水平，作为人类样本中 CDKAL1 酶活性的直接读出。
我们开发了一种基于定量 PCR（qPCR）的方法来测量 ms（2）修饰。tRNA（Lys）（UUU）使用 2 个独特的引物进行逆转录：反向引物 r1 设计为与该 tRNA 的中部退火，包括 A37 处的核苷酸，反向引物 r2 设计为与 A37 下游（3'）的区域退火。随后进行 qPCR 以检测相应的转录 cDNA。
tRNA（Lys）（UUU）的逆转录效率依赖于 ms（2）修饰。使用 r1 或 r2 引物获得的阈值循环数的相对差异精确地反映了 tRNA（Lys）（UUU）中 ms（2）修饰的水平，正如原始数学模型所预测的那样。该方法能够测量从人外周血样本中分离的总 RNA 中的 tRNA（Lys）（UUU）中的 ms（2）修饰水平，表明携带与 T2D 相关的 CDKAL1 基因型的个体中 tRNA（Lys）（UUU）的 ms（2）修饰率降低。此外，ms（2）修饰水平与胰岛素分泌相关。
结果表明 ms（2）修饰在 T2D 中的关键作用，并指出评估 T2D 风险的简单高通量方法的潜在临床用途。
