Abstract
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.
Highlights
Determining the function of every single gene remains a challenging task for modern biology
We initially speculated that efficient incorporation of 4TU into RNA molecules would largely depend on the following parameters: (i) efficiency of intra-cellular import of exogenous nucleobase, (ii) presence of enzyme(s) allowing conversion of the nucleobase into nucleotide tri-phosphate, (iii) and the existence of defined cultivation procedures where the nucleobase can be exogenously added
Given the known advantages of the methodology to analyze RNA dynamics and, RNA-protein interactions (e.g., Hafner et al, 2010; Ascano et al, 2012; Castello et al, 2012; Swiatkowska et al, 2012; Tallafuss et al, 2014; Duffy et al, 2015; Hulscher et al, 2016), we are confident that our proof of principle analysis broadens the archaeal molecular biology toolkit and will stimulate deeper analysis of RNA dynamics and gene expression networks in archaea
Summary
Determining the function of every single gene remains a challenging task for modern biology. In the post-genomic era, several functional analysis strategies can be applied to unravel the information stored at the DNA level. Comprehensive functional analysis of individual genes contained in an individual genome still remains a daunting task (e.g., Bork et al, 1998; Niehrs and Pollet, 1999; Wu et al, 2002; Schlitt et al, 2003; Fraser and Marcotte, 2004). Complex phenotypic traits rely essentially on the establishment of highly cooperative and dynamic gene networks that can integrate the sum of functions of several individual gene products at a given time (e.g., Barabasi and Oltvai, 2004; Fraser and Marcotte, 2004). Despite full genome sequencing of numerous organisms, the function of many open reading frames remains poorly characterized
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