5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG 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.

Keywords: SHOT-RNA; tRNA; tRNA half; tRNAHisGUG; −1 nucleotide.

FIGURE 1.

Terminal nucleotide analyses of BT-474 5′-SHOT-RNA^HisGUG identified by cP-RNA-seq. (A) The cloverleaf secondary structure of the major isodecoder of human cyto tRNA^HisGUG encoded by nine genes (Supplemental Fig. S1) on the genome. Nucleotide positions (np) are indicated according to the tRNA nucleotide numbering systems (Sprinzl et al. 1998). The ANG-cleavage sites for SHOT-RNA production, predicted by the 3′-terminal position of 5′-SHOT-RNA^HisGUG, are indicated by arrowheads. Regions from which 5′-SHOT-RNA^HisGUG molecules were derived are shown in black; other regions are shown in gray. (B) Pie chart indicating the 3′-terminal position of 5′-SHOT-RNA^HisGUG. (C) The six 5′-terminal variations identified in 5′-SHOT-RNA^HisGUG. (D) Pie charts showing the 5′-terminal positions of 5′-SHOT-RNA^HisGUG.

FIGURE 2.

Primer extension assay to determine the 5′-terminal position of 5′-SHOT-RNA^HisGUG. (A) The cloverleaf secondary structure of 5′-SHOT-RNA^HisGUG used as a primer extension template. The 5′-end-labeled 20-nt primer, which was hybridized to the D-arm of tRNA, is shown as a black solid line; nascent cDNA synthesized from the primer is indicated as a gray dotted line. Reverse transcription from the primer terminates at np 1 or −1 to yield a cDNA band with a length of 25 or 26 nt, respectively. (B) Synthetic mature tRNA^HisGUG containing either G₁ or G₋₁, or a 30- to 50-nt small RNA fraction of BT-474 cells were subjected to a primer extension assay for an analysis of the 5′-terminal position of 5′-SHOT-RNA^HisGUG. An assay without template RNA was also performed as a negative control experiment.

FIGURE 3.

TaqMan qRT-PCR method to analyze the 5′-terminal nucleotide of mature tRNA^HisGUG. (A) Schematic representation of the TaqMan qRT-PCR analysis used to quantify each 5′-terminal variant of mature tRNA^HisGUG. (B) Sequences and/or positions of the mature tRNA^HisGUG and the following TaqMan qPCR components: adapter, AS-disrupter, primers, and TaqMan probe. (C) Under the indicated conditions, this method was applied to synthetic mature tRNA^HisGUG containing G₋₁. The reaction containing only T4 RNA ligase (far left) was set to one, and fold changes relative to this reference are shown; bars indicate SD from three independent experiments. Amplified cDNA bands observed in native PAGE after 40 cycles of PCR are also shown. (D) Synthetic mature tRNAs^HisGUG starting from G₁, G₋₁, and U₋₁ were mixed at the indicated ratios and quantified by the TaqMan qRT-PCR. Detected amounts were calculated using standard curves (Supplemental Fig. S5), and the relative abundances of detected tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals.

FIGURE 4.

Variations in 5′-terminal nucleotide from mature tRNA^HisGUG expressed in BT-474 cells. (A) Of note, 70- to 90-nt mature tRNA fractions of BT-474 cells were subjected to TaqMan qRT-PCR quantification of each 5′-terminal variant of mature tRNA^HisGUG. Expression levels were estimated using standard curves from synthetic tRNAs (Supplemental Fig. S5), and the relative abundances of mature tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals. (B) A primer extension assay to analyze the 5′-terminal positions of mature tRNA^HisGUG was performed using the 70- to 90-nt mature tRNA fraction from BT-474 cells.