ALKBH1 is a 2-oxoglutarate- and Fe2+-dependent dioxygenase responsible for multiple cellular functions. Here, we show that ALKBH1 is involved in biogenesis of 5-hydroxymethyl-2΄-O-methylcytidine (hm5Cm) and 5-formyl-2΄-O-methylcytidine (f5Cm) at the first position (position 34) of anticodon in cytoplasmic tRNALeu, as well as f5C at the same position in mitochondrial tRNAMet. Because f5C34 of mitochondrial tRNAMet is essential for translation of AUA, a non-universal codon in mammalian mitochondria, ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f5C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. We reconstituted formation of f5C34 on mitochondrial tRNAMetin vitro, and found that ALKBH1 first hydroxylated m5C34 to form hm5C34, and then oxidized hm5C34 to form f5C34. Moreover, we found that the frequency of 1-methyladenosine (m1A) in two mitochondrial tRNAs increased in ALKBH1-knockout cells, indicating that ALKBH1 also has demethylation activity toward m1A in mt-tRNAs. Based on these results, we conclude that nuclear and mitochondrial ALKBH1 play distinct roles in tRNA modification.

FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2′-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient–derived cells. Loss of 2′-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

Background. The authors analyzed the clinical usefulness of glutathione‐S‐transferase‐π (GST‐π) as a tumor marker in patients with oral cancer. Methods. GST‐π levels in plasma of 61 patients with oral squamous cell carcinomas, 65 patients with benign oral diseases, and 78 healthy subjects were investigated with the sandwich enzyme‐immunoassay (EIA) system. Results. Elevated GST‐π levels in plasma were observed in patients with oral cancer, but patients with benign oral diseases had normal GST‐π levels. More than 70% of patients with Stage III or IV oral cancer and more than 50% of those with Stage I and II disease had elevated levels of GST‐π in plasma. Elevated levels of GST‐π in plasma were also discovered in most patients with tumor recurring after surgery before recurrence was detected clinically. GST‐π also was found to be a useful marker for evaluating the response to chemotherapy, for monitoring postoperative tumor resectability or tumor burden, and for predicting the recurrence of tumor in patients with oral cancer. Conclusions. GST‐π is considered to be a useful aid for early diagnosis, predicting tumor extent, and determining parameters of treatment efficacy and prognosis for oral cancer.

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.

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