Abstract
Similar to DNA replication, translation of the genetic code by the ribosome is hypothesized to be exceptionally sensitive to small chemical changes to its template mRNA. Here we show that the addition of common alkylating agents to growing cultures of Escherichia coli leads to the accumulation of several adducts within RNA, including N(1)-methyladenosine (m1A). As expected, the introduction of m1A to model mRNAs was found to reduce the rate of peptide bond formation by three orders of magnitude in a well-defined in vitro system. These observations suggest that alkylative stress is likely to stall translation in vivo and necessitates the activation of ribosome-rescue pathways. Indeed, the addition of alkylation agents was found to robustly activate the transfer-messenger RNA system, even when transcription was inhibited. Our findings suggest that bacteria carefully monitor the chemical integrity of their mRNA and they evolved rescue pathways to cope with its effect on translation.
Highlights
Nucleic acids are consistently experiencing damage from numerous endogenous and exogenous insults, including reactive-oxygen species, ultraviolet radiation, and alkylating agents (Wurtmann and Wolin, 2009; Simms and Zaher, 2016; Yan and Zaher, 2019a)
We have recently shown that the addition of methyl methanesulfonate (MMS) to growing yeast cultures leads to the accumulation of alkylation adducts in RNA (Yan et al, 2019b)
Nucleosides were generated by incubating the P1-reaction products with calf-intestinal phosphatase (CIP); and analyzed by liquid chromatography – mass spectrometry (LC-MS)
Summary
Nucleic acids are consistently experiencing damage from numerous endogenous and exogenous insults, including reactive-oxygen species, ultraviolet radiation, and alkylating agents (Wurtmann and Wolin, 2009; Simms and Zaher, 2016; Yan and Zaher, 2019a). For example, the addition of alkylating compounds was found to significantly increase the levels of m1A within mRNA, among several other adducts (Yan et al, 2019b) These compounds were observed to activate eukaryotic-quality-control pathways known to be responsible for ribosome rescue, suggesting that alkylation stress causes ribosome stalling presumably due to its damaging effect on mRNA. Alkylation stress is likely to significantly impact translation through its effect on mRNA and rescue pathways such as trans translation are responsible for dealing with its consequences Consistent with these proposals, E. coli strains lacking functional tmRNA were found to be sensitive to alkylating agents and exhibited delayed recovery compared to wild-type (WT) cells. Our data suggest chemical damage to mRNA is highly detrimental to cellular homeostasis, even in organisms where mRNA is highly transient
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