tRNA Biology in the Pathogenesis of Diabetes: Role of Genetic and Environmental Factors

Abstract

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes.

Keywords: insulin resistance; obesity; pancreatic β-cells; tRNA; tRNA fragments; tRNA modifications; type 2 diabetes.

Figure 1

Cloverleaf 2D tRNA structure, tRNA modifications by TRMT10A, CDKAL1, and CDK5RAP1, tRNA aminoaylation, and biogenesis of tRNA fragments. Typical cloverleaf tRNAs structure of a cytosolic tRNA showing the D-loop, the T loop, the variable loop, and the anticodon loop. Mature tRNAs containing the CCA sequence in their 3′-end are covalently charged with their corresponding aminoacid a reaction catalysed by the aminoacyl-tRNA synthetases (aaRSs). tRNA molecules are postranscriptionally modified by tRNA modifying enzymes. TRMT10A is an S-adenosyl-L-methionine-dependent guanine N¹-methyltransferase that catalyses the formation of N¹-methylguanine at guanosine in position 9 of cytosolic tRNAs (m¹ G₉). Mutations in TRMT10A cause young onset diabetes and microcephaly. CDKAL1 catalyses the methylthiolation of N⁶-threonyl carbamoyladenosine (t⁶A), leading to the formation of 2-methylthio-N⁶-threonylcarbamoyladenosine (ms ²t⁶A₃₇) at adenosine in position 37 of tRNA^Lys_(UUU). Intronic polymorphisms in CDKAL1 increase T2D risk. CDK5RAP1 is a mitochondrial tRNA methylthiotransferase that catalyses the conversion of N⁶-(dimethylallyl)adenosine (i⁶A) to 2-methylthio-N⁶-(dimethylallyl)adenosine (ms ²i⁶A₃₇) at adenosine in position 37 of different mt-tRNAs. Impaired CDK5RAP1 function has been associated with Maternal Inherited Diabetes and Deafness (MIDD) and Mitochondrial Encephalopathy, Lactic acidosis, and Stroke-like episodes. tRNA cleavage by Dicer, angiogenin, and other still unidentified endonucleases can give rise to tRNA fragments. Mature tRNAs are cleaved by DICER at the D-loop and T loop to produce 5′- and 3′- small tRNA fragments (5′-tRFs and 3′-tRFs), respectively. Angiogenin cleaves mature tRNA at the anticodon-loop to produce 5′ and 3′ tRNA halves (tRHs). Internal tRNA fragments (i-tRFs) comprise the anticodon- loop of the tRNA and are generated by unknown RNases. Immature (precursor) tRNAs having the 5′ leader (purple) and 3′ trailer (red) sequences are cleaved by RNaseP and RNase Z/ELAC2 generating 5′U-tRF and tRFs-1, respectively.

Figure 2

Genetic and environmental inhibition of tRNA-modifying enzymes. Genetic variants in CDKAL1 have been associated with increased T2D risk. Iron deficiency and oxidative stress (induced by HFD or mitochondrial dysfunction) impair the activity of both CDKAL1 and its mitochondrial homologue CDK5RAP1 by reducing the availability of 4Fe-4S clusters and CysSSH (both needed for their catalytic activity). Impaired CDKAL1 activity results in reduced ms² modification in cytosolic tRNA^Lys. This causes impaired tRNA^Lys incorporation into proteins (mainly proinsulin), reduced proinsulin processing, proinsulin accumulation in the ER that causes ER stress, and decreased insulin secretion. At the mitochondrial level, impaired CDK5RAP1 function leads to reduced ms² modification in mt-tRNAs resulting in impaired mitochondrial protein synthesis. This results in mitochondrial dysfunction and oxidative stress that may amplify CDK5RAP1 failure.

Figure 3

The saturated free fatty acid palmitate impairs CDKAL1-mediated ms² modification in cytosolic tRNA^Lys_(UUU). Human islets from seven nondiabetic organ donors were exposed or not (CT) for 48 h to 0.5 mM palmitate (PAL) in culture medium containing 1% BSA and no serum. Total tRNA was extracted and RNA^Lys ms² was analysed by real-time PCR as described by Xie and colleagues [114]. (A) Representative scheme of the assay (image adapted from Xie et al. [114]). For assessing tRNA^Lys ms² modification at A₃₇, total RNA is reversed transcribed using Rev1 or Rev2 primers targeting human tRNA^Lys. The cDNA obtained is then used as template for real-time PCR using the primer combinations (Fw1 + Rev1) or (Fw1 + Rev2). Xie et al. have shown that the ms² modification blocks the reverse transcription. Thus, the presence of the ms² modification impairs cDNA production when the Rev2 primer is used but it does not alter the reverse transcription with Rev1. (B) tRNA^Lys ms² in human islets. The results (means ± SE) are expressed as 2^-ΔΔCt. ΔCt = (Ct^Rev2-Fw1 − Ct^Rev1-Fw1). High 2^-ΔΔCt indicates reduced ms² modification. This data indicates that PAL exposure reduces CDKAL1-mediated tRNA^Lys modification in human islets. (C) RNA sequencing data of CDKAL1 expression (expressed in RPKM, means ± SE, from Cnop et al.) of five human islet preparations exposed or not (CT) for 48h to PAL, showing that PAL exposure does not affect CDKAL1 expression. Data points represent independent human islet preparations.

Figure 4

Impact of hight fat diet-mediated tRNA fragmentation in intergenerational inheritance of metabolic traits. High fat feeding in males leads to increased DNMT2-mediated m⁵C and m⁵G tRNA methylation and fragmentation in epidydimal and/or prostate acinar cells. The tRNA fragments generated (essentially 5′-tRHs) are transferred to sperm and transmitted to offspring upon fecundation affecting the metabolic health of F1 and F2 generations which show obesity, insulin resistance, mild glucose intolerance and reduced β-cell mass. Direct microinjection of sperm RNA fractions containing the 5′ tRHs into zygotes derived from normal chow-fed parents reproduced the metabolic phenotype in the offspring.