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Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNA HisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G -1 ) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNA HisGUG containing G -1 , U -1 , A -1 , or C -1 or lacking the -1 nucleotide (starting from G 1 ). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNA HisGUG containing U -1 as well as the one containing G -1 Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNA HisGUG variants. A sequence library of sex hormone-induced 5'-tRNA halves (5'-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5'-tRNA HisGUG halves containing G -1 , U -1 , or G 1 as 5'-terminal nucleotides. Although the detected 5'-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5'-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5'-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNA HisGUG molecules and provide insights into tRNA HisGUG maturation and the regulation of tRNA half production.

5′-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5′-halves

5′-Terminal nucleotide variations in human cytoplasmic tRNA^HisGUG and its 5′-halves