This work was supported by the National Institutes of Health (NIH) under grant R01 GM70641 to Valérie de Crécy- Lagard, partially under grant AI118999 to Kelly C. Rice, and the Florida Education Fund McKnight Doctoral Fellowship awarded to Silvia S. Orsini.
RNA -- Metabolism, Genetic regulation, Nucleosides -- Synthesis, Adenosine -- Detection
Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALys UUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared to WT). Moreover, mutants in two other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNAs levels in S. mutans suggest they may be regulated by growth phase.
Bacusmo, J. M., Orsini, S. S., Hu, J., DeMott, M., Thiaville, P. C., Elfarash, A., ... & Iwata-Reuyl, D. (2017). The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans. RNA biology, 1-10.