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, tRNAHisGUG 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 tRNAHisGUG 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 tRNAHisGUG 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'-tRNAHisGUG 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'-tRNAHisGUG 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 tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.