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 has been known to uniquely contain an additional guanosine residue at the -1 position (G) 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 containing G, U, A, or C or lacking the -1 nucleotide (starting from G). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNA containing U as well as the one containing G Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNA 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 halves containing G, U, or G 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 molecules and provide insights into tRNA maturation and the regulation of tRNA half production.

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease

“…S4C and S4D ). The predominant 3′-end of 5′-HisGUG was G34, consistent with our previous findings in human cell lines and mouse tissues 24, 30, 36, 40 .…”

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease

“…S4C and S4D ). The predominant 3′-end of 5′-HisGUG was G34, consistent with our previous findings in human cell lines and mouse tissues 24, 30, 36, 40 .…”

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

“…In contrast to previous reports describing the 5′-terminal G −1 as a major presence in tRNA HisGUG , the identified 5′-terminal variations of 5′-half HisGUG revealed the substantial presence of the U −1 residue (Figure 4 ), which is consistent with our recent study on 5′-half HisGUG in human cancer cells ( 53 ). The major presence of the U −1 residue in td-piR HisGUG (Figure 4 ) should be attributable to their selective loading onto Piwi proteins.…”

Immunostimulatory short non-coding RNAs in the circulation of patients with tuberculosis infection

“…Moreover, a substantial portion of the 5′-halves HisGUG lack G –1 and instead have G +1 as the 5′-terminal nucleotide in both human cell lines and mouse tissues. 34 , 44 , 50 , 51 Consistent with these findings, all abundant circulating 5′-tRFs and 5′ halves of tRNA HisGUG in the plasma of Mtb-infected patients lacked the G –1 nucleotide ( Figure 3 A). Analyses encompassing all circulating 5′-tRFs and 5′ halves of tRNA HisGUG showed that, in healthy samples, approximately 89% of these sncRNAs were missing the G –1 nucleotide ( Figure 3 C).…”

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease

“…S4C and S4D ). The predominant 3′-end of 5′-HisGUG was G34, consistent with our previous findings in human cell lines and mouse tissues 24, 30, 36, 40 .…”

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

“…In contrast to previous reports describing the 5′-terminal G −1 as a major presence in tRNA HisGUG , the identified 5′-terminal variations of 5′-half HisGUG revealed the substantial presence of the U −1 residue (Figure 4 ), which is consistent with our recent study on 5′-half HisGUG in human cancer cells ( 53 ). The major presence of the U −1 residue in td-piR HisGUG (Figure 4 ) should be attributable to their selective loading onto Piwi proteins.…”

Immunostimulatory short non-coding RNAs in the circulation of patients with tuberculosis infection

“…Moreover, a substantial portion of the 5′-halves HisGUG lack G –1 and instead have G +1 as the 5′-terminal nucleotide in both human cell lines and mouse tissues. 34 , 44 , 50 , 51 Consistent with these findings, all abundant circulating 5′-tRFs and 5′ halves of tRNA HisGUG in the plasma of Mtb-infected patients lacked the G –1 nucleotide ( Figure 3 A). Analyses encompassing all circulating 5′-tRFs and 5′ halves of tRNA HisGUG showed that, in healthy samples, approximately 89% of these sncRNAs were missing the G –1 nucleotide ( Figure 3 C).…”

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease

“…S4C and S4D ). The predominant 3′-end of 5′-HisGUG was G34, consistent with our previous findings in human cell lines and mouse tissues 24, 30, 36, 40 .…”

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

“…In contrast to previous reports describing the 5′-terminal G −1 as a major presence in tRNA HisGUG , the identified 5′-terminal variations of 5′-half HisGUG revealed the substantial presence of the U −1 residue (Figure 4 ), which is consistent with our recent study on 5′-half HisGUG in human cancer cells ( 53 ). The major presence of the U −1 residue in td-piR HisGUG (Figure 4 ) should be attributable to their selective loading onto Piwi proteins.…”

Immunostimulatory short non-coding RNAs in the circulation of patients with tuberculosis infection

“…Moreover, a substantial portion of the 5′-halves HisGUG lack G –1 and instead have G +1 as the 5′-terminal nucleotide in both human cell lines and mouse tissues. 34 , 44 , 50 , 51 Consistent with these findings, all abundant circulating 5′-tRFs and 5′ halves of tRNA HisGUG in the plasma of Mtb-infected patients lacked the G –1 nucleotide ( Figure 3 A). Analyses encompassing all circulating 5′-tRFs and 5′ halves of tRNA HisGUG showed that, in healthy samples, approximately 89% of these sncRNAs were missing the G –1 nucleotide ( Figure 3 C).…”