A selective and sensitive detection system for 4-thiouridine modification in RNA

Abstract

4-Thiouridine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s4U in tRNA are ongoing in the tRNA modification field. s4U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s4U is essential for progress in the fields of RNA technologies and tRNA modification. Here, we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s4U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation; however, addition of a denaturation step improves the limit of detection.Thermus thermophilustRNAs, which abundantly contain 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s4U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from aT. thermophilustRNA 4-thiouridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s4U detection system is that MTSEA biotin-XX reacts only with s4U and not with other sulfur-containing modified nucleosides such as s2U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s4U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s4U detection system can also be used for the analysis of s4U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs inS. cerevisiaecells.