Abstract
Epitranscriptomic marks, in the form of enzyme catalyzed RNA modifications, play important gene regulatory roles in response to environmental and physiological conditions. However, little is known with respect to how acute toxic doses of pharmaceuticals influence the epitranscriptome. Here we define how acetaminophen (APAP) induces epitranscriptomic reprogramming and how the writer Alkylation Repair Homolog 8 (Alkbh8) plays a key gene regulatory role in the response. Alkbh8 modifies tRNA selenocysteine (tRNASec) to translationally regulate the production of glutathione peroxidases (Gpx’s) and other selenoproteins, with Gpx enzymes known to play protective roles during APAP toxicity. We demonstrate that APAP increases toxicity and markers of damage, and decreases selenoprotein levels in Alkbh8 deficient mouse livers, when compared to wildtype. APAP also promotes large scale reprogramming of many RNA marks comprising the liver tRNA epitranscriptome including: 5-methoxycarbonylmethyluridine (mcm5U), isopentenyladenosine (i6A), pseudouridine (Ψ), and 1-methyladenosine (m1A) modifications linked to tRNASec and many other tRNA’s. Alkbh8 deficiency also leads to wide-spread epitranscriptomic dysregulation in response to APAP, demonstrating that a single writer defect can promote downstream changes to a large spectrum of RNA modifications. Our study highlights the importance of RNA modifications and translational responses to APAP, identifies writers as key modulators of stress responses in vivo and supports the idea that the epitranscriptome may play important roles in responses to pharmaceuticals.
Highlights
Epitranscriptomic marks in the form of enzyme-catalyzed RNA modifications are important gene regulatory signals, with defects linked to disrupted gene expression, disease onset and disease progression [1]
Glutathione peroxidase 3 (Gpx3) is an extracellular antioxidant enzyme that aids in scavenging hydrogen peroxide as well as other hydroperoxides [50]
In response to APAP, there is a significant increase (p = 0.02) in Gpx3 expression in WT compared to its saline control, and this increased response was not observed in Alkbh8Def liver tissue (Figure 1D)
Summary
Epitranscriptomic marks in the form of enzyme-catalyzed RNA modifications are important gene regulatory signals, with defects linked to disrupted gene expression, disease onset and disease progression [1]. The location and levels of specific RNA modifications have been shown to drive gene expression. Chemical modifications on tRNAs provide regulation of structure and function, while those occurring in the anticodon loop positions 34 to 37 can regulate translation and fidelity. The wobble position (34) in the anticodon stem loop of tRNA allows pairing to occur with more than one nucleoside, allowing for a single tRNA to decode multiple codons with different 3 nucleosides [10,11,12]. Wobble position modifications to uridine (U), cytidine (C), guanosine (G), and adenosine (A) have all been reported and can include simple methylations to more elaborate chemical additions, which can regulate anticodon-codon interactions while preventing translational errors [10,11]
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