Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice
Fan-Yan Wei, … , Tsutomu Suzuki, Kazuhito Tomizawa
Fan-Yan Wei, … , Tsutomu Suzuki, Kazuhito Tomizawa
Published August 15, 2011
Citation Information: J Clin Invest. 2011;121(9):3598-3608. https://doi.org/10.1172/JCI58056.
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Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice

Abstract

The worldwide prevalence of type 2 diabetes (T2D), which is caused by a combination of environmental and genetic factors, is increasing. With regard to genetic factors, variations in the gene encoding Cdk5 regulatory associated protein 1–like 1 (Cdkal1) have been associated with an impaired insulin response and increased risk of T2D across different ethnic populations, but the molecular function of this protein has not been characterized. Here, we show that Cdkal1 is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNALys(UUU) and that it is required for the accurate translation of AAA and AAG codons. Mice with pancreatic β cell–specific KO of Cdkal1 (referred to herein as β cell KO mice) showed pancreatic islet hypertrophy, a decrease in insulin secretion, and impaired blood glucose control. In Cdkal1-deficient β cells, misreading of Lys codon in proinsulin occurred, resulting in a reduction of glucose-stimulated proinsulin synthesis. Moreover, expression of ER stress–related genes was upregulated in these cells, and abnormally structured ER was observed. Further, the β cell KO mice were hypersensitive to high fat diet–induced ER stress. These findings suggest that glucose-stimulated translation of proinsulin may require fully modified tRNALys(UUU), which could potentially explain the molecular pathogenesis of T2D in patients carrying cdkal1 risk alleles.

Authors

Fan-Yan Wei, Takeo Suzuki, Sayaka Watanabe, Satoshi Kimura, Taku Kaitsuka, Atsushi Fujimura, Hideki Matsui, Mohamed Atta, Hiroyuki Michiue, Marc Fontecave, Kazuya Yamagata, Tsutomu Suzuki, Kazuhito Tomizawa

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Figure 1

Methylthiolation of tRNALys(UUU) by Cdkal1 controls the decoding accuracy of the lysine codon.

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Methylthiolation of tRNALys(UUU) by Cdkal1 controls the decoding accurac...
(A) The molecular structure of tRNALys(UUU) and ms2t6A. (B) Results of a mass spectrometric analysis of the ms2t6A modification of tRNA in MIN6 and HeLa cells. The upper panels show the UV trace, and the middle and lower panels show the mass chromatograms for detecting t6A (m/z 413, arrow) and ms2t6A (m/z 459, arrow), respectively. (C) Results of a mass spectrometric analysis of the ms2t6A modification of tRNA isolated from the pancreas of Cdkal1–/– and WT mice. The arrow indicates ms2t6A (m/z 459). (D) Modification of tRNALys(UUU) isolated from the liver of Cdkal1–/– and WT mice. The upper panels show mass chromatograms of GGDp fragments in tRNALys(UUU). The middle and lower panels show mass chromatograms of ms2t6AAΨp fragments and t6AAΨp fragments, respectively. (E) WT and ΔyqeV cells were transformed with a reporter plasmid in which both Renilla renilla and firefly luciferases are cloned with the lac promoter (upper panel). Relative activity was determined by normalizing firefly luciferase intensity to renilla luciferase intensity (F/R, lower panel). Data are presented as the mean ± SEM, and asterisks indicate statistical significance determined by Student’s t test. ***P < 0.001; n = 4. (F) The expression level of the fusion protein of firefly and renilla luciferase after IPTG treatment induction was determined in WT and ΔyqeV cells (E) by Western blot.