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

Abstract

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.