Defective i6A37 Modification of Mitochondrial and Cytosolic tRNAs Results from Pathogenic Mutations in TRIT1 and Its Substrate tRNA

Abstract

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA

“…By this assay, an oligo-DNA probe complementary to the anticodon loop (ACL) hybridizes better in the absence of i6A37 than in its presence; the ACL probe signal is internally controlled by a probe to a different region of the same tRNA, the T stem-loop (TSL probe) (Lamichhane et al 2011(Lamichhane et al , 2013aYarham et al 2014). Because the TSL probe serves as a quantitative internal control on the same tRNA, its signal intensity can be used to calibrate and measure differences in i6A37 modification efficiency (Lamichhane et al 2011(Lamichhane et al , 2013aYarham et al 2014). Accordingly, Figure 1C shows less ACL hybridization for cy-tRNA Ser in yYH1 (tit1 + , lane 1) relative to yNB5 (tit1-Δ, lane 2), while the same tRNA shows near equal TSL probe signal in the two strains, as expected (Fig.…”

“…A mutation in the human IPTase, TRIT1 and resultant i6A37 hypomodification of cy-and mt-tRNAs causes a disease phenotype characteristic of mitochondrial translation deficiency similar to other mitochondrial diseases attributed to defects in mt-tRNAs (Yarham et al 2014). Deletion of the S. pombe IPTase, Tit1, causes general decrease in cytoplasmic translation of mRNAs enriched with i6A37-cognate codons such as for ribosome subunits and translation factors (Lamichhane et al 2013a).…”

“…tit1-Δ cells also exhibit slow growth when the carbon nutrient is glycerol (Lamichhane et al 2013a). This glycerol phenotype as well as the two other tit1-Δ phenotypes is rescued by ectopic expression of S. pombe Tit1, human TRIT1 or the S. cerevisiae IPTase, Mod5 (Lamichhane et al 2011(Lamichhane et al , 2013aYarham et al 2014). Slow growth of S. pombe in glycerol sometimes reflects mitochondrial dysfunction (Boutry et al 1984) which if so in this case, tit1-Δ cells might provide a model system for the study of human TRIT1 mt-tRNA-i6A37 function.…”

“…Many nucleotides in tRNAs are post-transcriptionally modified and many affect tRNA stability or activity (Phizicky and Hopper 2010). A large number of mitochondrial disorders are due to mutations to mt-tRNAs encoded in the mt-DNA or to mt-tRNA modification or processing enzymes encoded in the nuclear DNA (Yarham et al 2010(Yarham et al , 2014Abbott et al 2014).…”

Protein folding and tRNA biology

“…The PHA6 northern blot assay is based on a DNA probe complementary to the anticodon loop which hybridizes better in the absence of i6A37 and a probe complementary to a different region of the same tRNA as a quantitative internal control. The procedure can be used to calibrate and measure differences in i6A37 modification (Lamichhane et al 2011(Lamichhane et al , 2013a(Lamichhane et al , b, 2016Yarham et al 2014).…”