A 2020 publication in Nucleic Acids Research 1 includes the following passage:
A variety of nucleoside analogs have been developed for metabolic RNA labeling in various eukaryotic cells (9–16). Among them, 4-thiouridine (4SU) and 5-ethynyluridine (EU) are two most widely used noncanonical nucleosides that can be conjugated via thiol coupling chemistry and click chemistry, respectively (9,10). Although the metabolic RNA labeling technique has been instrumental for studying RNA dynamics in eukaryotes, it is not applicable to bacteria.
The authors give no citations or further explanations for the bolded claim. Indeed, I have found at least one publication that contradicts this statement,2 showing 4SU labeling in E. coli:
We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine.
One explanation is that 4SU is a natural RNA modification in bacteria, as Rogers et al. 3 states that most bacterial tRNAs contain 4SU. So, while bacterial RNA can be labeled with exogenous 4SU, naturally occurring 4SU confounds analysis. Concerning EU, Blenkiron et al. 4 used this analog to label nascent RNAs from uropathogenic E. coli in order to track the fate of bacterial RNAs in membrane vesicles:
We then looked at whether we could track uptake of MV RNA cargo into treated host cells by labelling UPEC nascent RNA in the bacteria before it was packaged with MVs. This was achieved by adding an excess of a modified uracil, 5EU (5-ethynyl uridine), which was incorporated into the newly synthesised and MV associated RNA.
Additionally, the text of this research grant award abstract 5 suggests that EU is suitable for labeling both E. coli and B. subtilis:
We have been investigating whether bacteria can incorporate the “clickable” RNA analog 5-ethynyl uridine (EU) for subsequent attachment of fluorescent label (Alexa Fluor) for microscopic detection or a chemical tag (desthiobiotin) for streptavidin-mediated RNA capture. The results for both aspects so far are a qualified “yes”. Both Escherichia coli and Bacillus subtilis grown with EU can subsequently be detected by fluorescence microscopy, but E. coli becomes considerably more brightly labeled.
I can find no research stating EU / 5EU naturally occurs in bacterial RNA.
My question, in short: What aspects of bacterial metabolism or RNA composition justify the claim that 4SU and EU are "not applicable" for bacterial RNA labeling?
Edit, 1 March 2021 -- I'm adding a bounty to garner more attention. From further reading, I suspect the answer to be one of 3 possibilities.
- Per David's comment, the bacterial cell wall may be impermeable to certain formulations of nucleoside analogs or the reagents subsequently used in thiol coupling / click chemistry.
- Certain nucleoside analogs are toxic in bacteria to an extent not seen in eukaryotic cells, perhaps by a mechanism that causes the elongating RNA polymerase to stall.
- 4SU and EU labeling are applicable to bacteria, and the claims of the authors are unsubstantiated.
- Meng L, Guo Y, Tang Q, Huang R, Xie Y, Chen X. Metabolic RNA labeling for probing RNA dynamics in bacteria. Nucleic Acids Res. 2020 Dec 16;48(22):12566-12576.
- Favre A, Bezerra R, Hajnsdorf E, Lemaigre Dubreuil Y, Expert-Bezançon A. Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis. Eur J Biochem. 1986 Nov 3;160(3):441-9.
- Rogers KC, Crescenzo AT, Söll D. Aminoacylation of transfer RNAs with 2-thiouridine derivatives in the wobble position of the anticodon. Biochimie. 1995;77(1-2):66-74.
- Blenkiron C, Simonov D, Muthukaruppan A, Tsai P, Dauros P, Green S, Hong J, Print CG, Swift S, Phillips AR. Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA. PLoS One. 2016 Aug 8;11(8):e0160440.
- Click-chemistry labeling of RNA: A new tool for microbial ecology?