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

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 G₁). 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 G₁ 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.