Abstract
Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.
Highlights
Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA
We reasoned that if damaged mRNA is subject to no-go decay (NGD), deletion of XRN1 should increase the levels of modified nucleotides in the mRNA pool such as the oxidation product 8-oxoG
Total RNA and mRNA-enriched samples were treated with P1 nuclease and the resulting nucleotides were dephosphorylated to nucleosides using calf intestinal phosphatase (CIP) before analysis
Summary
Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. NGD and RQC have been studied almost exclusively in the context of artificial reporters that were genetically engineered to harbor roadblocks that stop ribosomes from translating These blocks include: stable-RNA-secondary structures such as long stem loops, internal polyA sequences, stretches of rare codons, inhibitory codon pairs, and sequences encoding peptides that interact with the exit tunnel of the ribosome[1,2,23,28,29,30]. Studies employing these reporters have been instrumental in unraveling the key mechanistic details related to NGD and RQC, the utility of these processes and their activation in response to different biological cues remain unclear. Some of these modifications, including N1-methyladenosine (m1A), N1-methylguanosine (m1G), and N3-methylcytosine (m3C), are cytotoxic when present in DNA as they block replication and are predicted to stall translation[38,40]
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