title: 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). 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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Molecular Physiology, Kumamoto University, Kumamoto, Japan;
authors: Peiyu
authors: P
authors: Xie
authors: Fan-Yan
authors: FY
authors: Wei
authors: Shoji
authors: S
authors: Hirata
authors: Taku
authors: T
authors: Kaitsuka
authors: Tsutomu
authors: T
authors: Suzuki
authors: Takeo
authors: T
authors: Suzuki
authors: Kazuhito
authors: K
authors: Tomizawa
mesh_terms: D000818:Animals; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D018570:Risk Assessment; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D012359:tRNA Methyltransferases
chemical_list: D009419:Nerve Tissue Proteins; D013438:Sulfhydryl Compounds; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; D051360:Cyclin-Dependent Kinase 5; C525110:CDKAL1 protein, human; C559068:CDKAL1 protein, mouse