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 (ms2) modification of the adenine at position 37 (A37) of cytoplasmic tRNALys(UUU). We investigated the ms2-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. tRNALys(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 ms2-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.

Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion

“…CBS and CTH were silenced by siRNAs, and the levels of ms 2 modification in cytosolic tRNA Lys(UUU) were examined using quantitative PCR (17). There was a significant decrease in the ms 2 levels in CBS- and CTH -silenced cells, when compared with control cells (Figure 2A).…”

“…The ms 2 modification levels were analyzed using a quantitative PCR-based method as described previously (17). The levels of ms 2 -modified tRNA Lys(UUU) were normalized to the total tRNA Lys(UUU) (17). In all experiments, the ms 2 modification levels in control cells are expressed as 100%.…”

Adaptive Human CDKAL1 Variants Underlie Hormonal Response Variations at the Enteroinsular Axis

“…Intronic CDKAL1 variants rs7754840, rs7756992, and rs10946398 have been associated with variations in insulin release, pancreatic cell functions, hemoglobin A1C level, and/or response to pancreatic KATP channel agonists [8], [38], [39] as well as type 2 diabetes, GDM, ulcerative colitis, Crohn's disease, obesity, and/or birth weight [9], [25], [26], [28]. CDKAL1 has been shown to function as a tRNA modification enzyme, and its activity is associated with ATP generation and first-phase insulin secretion [40], [41], [42]. The carriage of CDKAL1 risk variants may lead to lower insulin release and impaired conversion of proinsulin to insulin in pancreatic beta cells [43], [44], [45].…”

Modify or die? - RNA modification defects in metazoans

“…38 Human carriers of TIID alleles show reduced levels of ms 2 t 6 A 37 , they fail to generate and to secrete insulin, and they may respond differently to pharmacological treatment of TIID. [40][41][42][43][44] Consistent with this, CDKAL1 alleles correlate strongly with an increased risk for coronary artery diseases, most likely as a downstream effect of TIID. 45 Furthermore, CDKAL1 was identified as a risk factor for Crohn's disease (CD) and for Psoriasis.…”