Epitranscriptomic Reprogramming Is Required to Prevent Stress and Damage from Acetaminophen

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 (tRNA^Sec) 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 tRNA^Sec 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.

Keywords: Alkbh8; RNA modification; acetaminophen; epitranscriptomic; stress response; tRNA.

Figure 1

Writer deficient mouse livers have increased sensitivity to APAP induced stress and display decreased selenoprotein levels. (A) APAP can be converted into a non-toxic form by UDP-glucuronosyltransferases (UGT) or a toxic form by cytochrome P450 enzyme (CYP2E1) to generate the reactive intermediate metabolite N-actyl-p-benzoquinone imine (NAPQI). Male C57Bl/6 WT (blue) and Alkbh8^Def (red) mice (8–12 weeks old) were exposed to a single dose of APAP (B–D) or daily dose of APAP over 4 days (E–G), and tissue/blood was harvested. (B,E) ALT and (C,F) 8-isoprostane levels were determined in blood and liver samples, respectively for WT saline (blue circle), Alkbh8^Def saline (red square), WT APAP (blue triangle) and Alkbh8^Def APAP (red triangle) (D,G) Gpx3 protein levels in the liver were evaluated using the ProteinSimple WES system. Statistical significance (N = 3) was measured by an unpaired t-test with (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 2

Next-generation mRNA sequencing analysis of the transcriptional response to APAP. WT and Alkbh8^Def mice (N = 3) were left untreated or exposed to a single dose of APAP and livers were harvested after 6 h and RNA was purified and subject to mRNA-seq. Enhanced volcano plots from mRNA-seq data were generated for each of the comparisons and metascape analysis were performed for log₂ FC > 2.0 and p_adj. values ≤ 0.05 for (A,B) WT APAP vs. WT Saline and (C,D) Alkbh8^Def APAP vs. Alkbh8^Def Saline.

Figure 3

WT and Alkbh8^Def livers have a similar transcriptional response to a single dose of APAP. mRNA-seq data for (A) Alkbh8^Def APAP vs. WT APAP was compared using an enhanced volcano plot. (B) Post-transcriptional regulation of translation by Alkbh8 and epitranscriptomic marks. Alkbh8 requires the small accessory protein Trm112 and S-adenosylmethionine (SAM) to modify the wobble position of tRNA^Sec (C) from cm⁵U to mcm⁵U, which is then further modified to the ribose-methylated derivative mcm⁵Um. (D) Stop-codon recoding utilizes mcm⁵U and mcm⁵Um in tRNA^Sec to decode UGA to produce selenoproteins. In addition to Alkbh8-catlyzed epitranscriptomic marks, elongation factors, a selenocysteine insertion sequence (SECIS), accessory proteins, and an internal UGA stop codon are used to promote the translation of selenocysteine containing proteins.

Figure 4

Multiple RNA modifications on tRNA for selenocysteine and other tRNAs are differentially regulated in WT and Alkbh8^Def livers in response to a single dose of APAP. (A) Epitranscriptomic marks found on tRNA^Sec. (B,C) WT and Alkbh8^Def mice (N = 3) were exposed to a single dose of APAP and livers were harvested 6 h after dosing. (B) Modifications that occur throughout tRNA^Sec and other tRNAs were measured using LC-MS/MS. (C) Writer enzyme gene count changes in expression vs. p-value determined. (D) Selenoprotein levels (N = 3) were evaluated using the ProteinSimple WES system. Statistical significance was determined using an unpaired t-test with (* p < 0.05, ** p < 0.01).

Figure 5

The epitranscriptome is differentially regulated in WT and Alkbh8^Def livers in response to a single dose of APAP. (A) 37 epitranscriptome marks were measured with LC-MS/MS and the log₂fold change was normalized relative to WT saline data and shown as a heat map. (B) Data for each modification under the four conditions was compared for statistical significance (N = 3) using an unpaired t-test with (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 6

Writer deficient mice have altered gene expression and response due to altered epitranscriptomic response. WT (N = 6) and Alkbh8^Def (N = 6) mice were IP injected daily with APAP. (A–C) Tissue was harvested at day 4 and RNA and protein were purified for analysis. (A,B) Enhanced volcano plots for mRNA-seq data were generated for each of the comparisons and metascape analysis was performed for log₂ FC > 2.0 and p_adj. values ≤ 0.05. (A) WT APAP vs. WT Saline and (B) Alkbh8^Def APAP vs. Alkbh8^Def Saline. (C) Selenoprotein levels were evaluated using the ProteinSimple WES system. Statistical significance (N = 3) was determined using an unpaired t-test with (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 7

Model of APAP sensitivity in writer and modification dysregulated Alkbh8^Def livers. Alkbh8 plays a protective role against oxidative stress-induced APAP toxicity. Alkbh8^Def mice experienced higher instances of oxidative stress, liver damage and overall decreased survival. The deficiency of Alkbh8 results in the dysregulation of the epitranscriptome and decreased selenoprotein expression.