Toward time-resolved analysis of RNA metabolism in archaea using 4-thiouracil

Archaea are widespread organisms colonizing almost every habitat on Earth. However, the molecular biology of archaea still remains relatively uncharacterized. RNA metabolism is a central cellular process, which has been extensively analyzed in both bacteria and eukarya. In contrast, analysis of RNA metabolism dynamic in archaea has been limited to date. To facilitate analysis of the RNA metabolism dynamic at a system-wide scale in archaea, we have established non-radioactive pulse labeling of RNA, using the nucleotide analog 4-thiouracil (4TU) in two commonly used model archaea: the halophile Euryarchaeota Haloferax volcanii, and the thermo-acidophile Crenarchaeota Sulfolobus acidocaldarius. In this work, we show that 4TU pulse labeling can be efficiently performed in these two organisms in a dose- and time-dependent manner. In addition, our results suggest that uracil prototrophy had no critical impact on the overall 4TU incorporation in RNA molecules. Accordingly, our work suggests that 4TU incorporation can be widely performed in archaea, thereby expanding the molecular toolkit to analyze archaeal gene expression network dynamic in unprecedented detail.