key: cord-0295286-kwb6atzg
authors: Evke, Sara; Melendez, J. Andres; Lin, Qishan; Begley, Thomas J.
title: Epitranscriptomic reprogramming is required to prevent stress and damage from acetaminophen
date: 2021-08-16
journal: bioRxiv
DOI: 10.1101/2021.08.16.456530
sha: 950c24e2e1ed0e9400e930f9ee53a4fd006a7a5e
doc_id: 295286
cord_uid: kwb6atzg

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 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 31 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 others. 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.

Epitranscriptomic marks catalyze RNA modifications and are important gene regulatory signals, with defects linked to disrupted gene expression, disease onset and disease progression (Kadumuri and Janga 2018) . Modifications on mRNA, rRNA and tRNA have been shown to be dynamically regulated to allow cells to survive different stressors and adapt to changes in cellular physiology (Cai et al. 2016; Chan et al. 2012; Endres, Dedon, and Begley 2015; Gu, Begley, and Dedon 2014) . In addition, the location and levels of specific RNA modifications have been shown to drive gene expression. tRNAs are the most heavily modified RNA species, and RNA modifications are critical for regulating tRNA structure, function and stability. Writer enzymes can catalyze or add tRNA modifications, while eraser enzymes can remove modifications and reader enzymes bind and recognize modifications. 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 (Agris, Vendeix, and Graham 2007) . 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 (Agris et al. 2017 (Agris et al. , 2018 Agris, Vendeix, and Graham 2007; Novoa, Pavon-eternod, and Pan 2012) .

While most mammalian tRNAs have 3 to 17 positions modified (Pan 2018) , the tRNA for selenocysteine (tRNA Sec ) is unique because it only has 4 positions modified. tRNA Sec modifications and writers include the 5-methoxycarbonylmethyluridine (mcm 5 U) and the ribosemethylated derivative, 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um) at position U34 which are dependent on Alkylation repair homolog 8 (Alkbh8) along with its accessory protein tRNA methyltransferase 112 (Trm112). The isopentenyladenosine (i 6 A) modification at position A37 is catalyzed by tRNA isopentenyltransferase 1 (Trit1) (Fradejas et al. 2013) , while the pseudouridine (Ψ) modification at position U55 is catalyzed by pseudouridine synthase 4 (Pus4) also known as TruB pseudouridine synthase family member 1 (Trub1) (Roovers et al. 2006) , and the 1-methyladenosine (m 1 A) modification at position A58 is catalyzed by the catalytic subunit of tRNA (adenine-N 1 -)-methyltransferase (Trmt61A) and RNA binding protein noncatalytic subunit tRNA (adenine (58)-N(1))-methyltransferase (Trm6) (M. Wang et al. 2016 ).

Alkbh8, mcm 5 U and mcm 5 Um have been shown to play a critical roles in the translation of selenoproteins Van Den Born et al. 2011; D. Fu et al. 2010; Pastore et al. 2012; Songe-Moller et al. 2010) . Selenoprotein synthesis is orchestrated by interactions between cis RNA elements and trans proteins and specifically modified tRNA to recode a UGA stop codon. Thus, the UGA selenocysteine (Sec) codon found within selenoprotein genes works with Alkbh8-modified tRNA, as well as other factors, to promote the translation of selenoproteins. There are 25 selenoproteins in human systems with rodents having only 24, with glutathione peroxidase 6 (Gpx6) being the discrepant selenoprotein (Regina et al. 2016) .

Some examples of selenoproteins include glutathione peroxidase (Gpx)1-4, thioredoxin reductases (TrxR) 1-3, selenoprotein S (SelS), selenoprotein K (SelK), and selenoprotein P (SelP).

Selenoproteins play roles in stress responses, regulating metabolism, immunity, and in embryonic vitality and development (Anouar et al. 2018; Hatfield et al. 2006; Hofstee, Cuffe, and Perkins 2020; Pappas, Zoidis, and Chadio 2019; Pitts et al. 2014; Rundlöf and Arner 2004; Shrimali et al. 2008 ). Many selenoproteins serve as antioxidant enzymes to mitigate damage caused by reactive oxygen species (ROS) (Arbogast and Ferreiro 2010; Benhar 2018; Chung et al. 2009; Couto, Wood, and Barber 2016; Eckers et al. 2013; Huang et al. 2020; Leonardi et al. 2019; Steinbrenner and Sies 2009; Zoidis et al. 2018) . A GeneTrap mouse deficient in Alkbh8 (Alkbh8 Def ) has been used to generate embryonic fibroblasts (MEFs) and these cells have been used to demonstrate that writer defects promote increased levels of intracellular ROS and DNA damage and reduced selenoprotein expression. Alkbh8 Def MEFS also display a proliferative defect, accelerate senescence and fail to increase selenoprotein levels in response hydrogen peroxide (H 2 O 2 ) (Endres et al. 2015a; Lee et al. 2020) . Alkbh8 Def mice also have increased lung inflammation and a disruption in lung glutathione levels (Leonardi et al. 2020) . Pulmonary exposure of Alkbh8 Def mice to naphthalene, the common polyaromatic hydrocarbon found in mothballs, increased stress markers and lung damage, when compared to their wildtype (WT) counterparts (Leonardi et al. 2020) .

Overall, these findings demonstrate that the epitranscriptomic writer Alkbh8 plays a vital role in protection from environmental stressors.

Little is known with respect to how the epitranscriptome responds to pharmaceutical stress. Acetaminophen (APAP) is one of the most prevalent over the counter drugs due to its analgesic and antipyretic properties and is consumed regularly by over 60 million Americans each week. APAP is a safe and effective drug that combats various ailments. However, in the United States nearly 50% of all cases of acute liver failure are due to an excessive use of APAP (Yoon et al. 2016 ). Gpx proteins have been linked to preventing APAP toxicity by providing protection from its metabolized forms (Mirochnitchenko et al. 1999) . The majority of APAP (85-90%) is acted upon by a phase II metabolism where APAP is chemically altered by UDPglucuronosyl transferases (Ugt) and sulfotransferases (Sult) and then converted to glucouronidated and sulfated metabolites that are eliminated in urine. A small amount (~2%) of APAP is excreted in urine without modification and the remaining is biometabolized by the cytochrome enzyme, Cyp2e1, which results in the highly reactive, toxic metabolite N-acetylpara-benzoquinone imine (NAPQI) (Mcgill and Jaeschke 2014) . Sufficient glutathione (GSH) in hepatocytes will reduce the reactive NAPQI and promote excretion in the urine. However excessive amounts of NAPQI can increase in oxidative stress, decrease GSH, and promote mitochondrial dysfunction leading to depletion of adenosine triphosphate (ATP) stores (Clark et al. 2012) .

Selenoproteins have been shown to provide protection from APAP toxicity.

Mirochnitchenko et al. demonstrated that glutathione peroxidase injection provided complete protection in mice administered an acute toxic dose of APAP (Mirochnitchenko et al. 1999) .

GPX3 overexpressing cells are more resistant to APAP toxicity than cells whose GPX3 levels were decreased by siGpx3 (Kanno et al. 2017) . Human GPX and mouse Gpx proteins are clearly linked to preventing APAP toxicity. As selenoprotein synthesis and activity are reliant on RNA modifications that drive selenocysteine utilization, we hypothesized that Alkbh8-regulated epitranscriptomic marks would be involved in the response to APAP. Here we test the hypothesis that the epitranscriptomic writer, Alkbh8 protects against APAP toxicity by catalyzing tRNA modifications that regulate the translation of stress response genes. We have phenotypically characterized liver tissue from both C57BL/6 WT and Alkbh8 Def treated mice with either saline (vehicle control) or toxic (600 mg/kg) APAP under both acute (6h) and sub-chronic (4 daily injections) exposure conditions. When challenged with APAP, WT liver tissue showed an increase in many selenoproteins whereas the Alkbh8 Def liver tissues were disrupted in their expression and show classic hall marks of stress and damage, including increased expression of the oxidative stress marker, 8-isoprostane, and liver damage marker, alanine transaminase (ALT). Transcriptional responses to acute APAP were similar between the genotypes; however, protein level differences were detected, which is indicative of post-transcriptional changes reflective of epitranscriptomic regulation. After a single dose of APAP, we have discovered that there is significant change in the levels of 31 tRNA modifications in response to APAP. Increases in the levels of tRNA modifications specific to tRNA Sec and many other tRNA isoacceptors was identified, with dysregulation of the many APAP dependent marks observed in Alkbh8 Def mice. Our study is one of the first to characterize the roles of epitranscriptomic marks and writers in response to APAP, and supports the idea that the epitranscriptome adapts to and modifies the effects of pharmaceuticals.

Animal studies were carried out in strict accordance with recommendations in the guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University at Albany Institutional Animal Care and Use Committee (Albany, NY), protocol #17-016/#20-014 for breeding and #18-010 for APAP exposure. Two homozygous strains of mice on the C57BL/6J background were bred, WT and Alkbh8 Def . Wild-type mice were purchased from Taconic Biosciences (Rensselaer, NY). Alkbh8 Def were produced through an insertional mutagenesis approach targeting the Alkbh8 gene in the parental E14Tg2a.4 129P2 embryonic stem cell line, as previously described (Endres et al., 2015) . Alkbh8 Def mice were produced using a vector containing a splice acceptor sequence upstream of a β -geo cassette (β-galactosidase/neomycin phosphotransferase fusion), which was inserted into intron 7 at chromosome position 9:3349468-3359589, creating a fusion transcript of Alkbh8-β-geo (Stryke et al. 2003) . Multiplex qRT-PCR and relative cycle threshold analysis (ΔΔCT) on genomic DNA derived from tail biopsies were used to determine animal zygosity for Alkbh8 Def , using TaqMan oligonucleosides specific for neomycin (Neo, target allele) and T-cell receptor delta (Tcrd, endogenous control). Neo forward ( For all animal studies, male mice between 8-12 weeks of age were used. Females were not used, as they have been shown to be less susceptible to APAP liver injury due to their accelerated recovery of hepatic glutathione (GSH) levels (Du et al. 2014) . Mice were sacrificed via CO 2 asphyxiation and in some cases, followed by non-survival vena cava puncture and necropsy of the liver. Whole blood was allowed to clot for 30 minutes at 25°C and organs were flash-frozen and placed in -80°C for storage.

APAP and Dulbecco's Phosphate Buffered Saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO). A 5 mg/mL (w/v) solution of APAP was made in a 15 mL conical tube with 10 mL of PBS and 0.5 g of APAP, due to lower solubility property of APAP, the solution was boiled until dissolved and then cooled to room temperature before injection. Injections for acute exposures occurred once and six hours after the exposure mice were sacrificed. For chronic conditions, injections were administered at the same time daily for four days, every 24 hours, and mice were sacrificed 24 hours post last injection. After injection, mice were observed up to one hour for recovery from anesthesia and to ensure mice were not experiencing any pain or distress. Mice were further monitored for signs of excess stress and weight loss, with any mouse losing more than 20% of its body weight sacrificed based on IACUC guidelines.

The Alanine Transaminase Colorimetric Activity Assay (Catalog no. 700260, Cayman

Chemical, Ann Arbor, MI) was performed following manufacturer's protocol to provide an indicator of liver damage. WT and Alkbh8 Def were exposed to either saline or APAP as described above and sacrificed via CO 2 asphyxiation. Cardiac puncture was then performed instructions, and absorbance was monitored at 340 nm over a period of 10 minutes at 1-minute intervals. Data analysis was performed and ALT activity was reported as U/L. Statistical significance was calculated utilizing three biological replicates for each exposure condition and genotype, with error bars denoting standard error of the mean and significance determined using Student's unpaired t-test in GraphPad Prism version 9.0 (GraphPad, San Diego, CA).

The 8-isoprostane assay (Catalog no. ab175819, Abcam, Cambridge, MA) was performed following manufacturer's protocol. Mice were exposed to either saline or APAP as described above and sacrificed via CO 2 asphyxiation. 1X Phosphate Buffered Saline (PBS) was injected into the portal vein and hepatic artery, to remove excess blood and other cellular debris from the liver. Once dissected from the mouse, the livers were washed in PBS and then stored at -80°C. Additional reagents that were used are 2N Sulfuric Acid Stop Solution (Catalog no.

DY994, R&D Systems, Minneapolis, MN), Triphenylphosphine (TPP) (Catalog no. T84409-1G

Sigma-Aldrich), and ethyl acetate (Catalog no. 270989, Sigma-Aldrich). 8-isoprostane was measured following manufacturer's protocol with the adjustment for low sample volume. Livers were weighed and homogenized in a 500 µL solution containing 5mg TPP in dH 2 O and then acidified by adding 4 µL of glacial acetic acid to ensure pH was ~ 4.0. Next, 500 µL of ethyl acetate was added to each sample, vortexed and centrifuged at 5,000 rpm for 5 minutes to separate the organic phase. The supernatant was collected and placed in a new conical tube and the previous step was performed one more time. The pooled organic phase supernatant was dried using nitrogen gas and then reconstituted in 20 µL 100% ethanol. Samples were further processed according to manufacturer's protocol and then used for ELISA measurement and analyzed. Statistical significance was calculated utilizing three biological replicates for each exposure condition and genotype with error bars denoting standard error of the mean and significance determined using Student's unpaired t-test in GraphPad Prism v9.

Primary antibodies used for quantification were as follows: 

Small RNAs were first isolated from liver tissue using PureLink™ miRNA Isolation Kit A method to extract peak areas from raw data to allow quantification was developed using a combination of instrument manufactures' suites, MassLynx V4.1 and TargetLynx (Waters, USA). These methods allowed extraction of information to produce calibration curves from each RNA modification standard. In addition, these programs were used to extract the peak areas to be extrapolated on the standard calibration curves for quantification of RNA modifications. In house Python script combined with Originlab software suite 2017 was used to quantify various RNA modifications. Heat maps were generated using MORPHEUS versatile matrix visualization and analysis software (https://software.broadinstitute.org/morpheus).

Statistical significance was calculated utilizing three biological replicates for each exposure condition and genotype. Statistical significance was determined using a student's unpaired ttest.

Total RNA was isolated from liver tissue. Samples were placed in 1 mL of Trizol reagent Gene count plots of epitranscriptomic writers were created using DeSeq2 and ggplot2 (Wickham 2016) packages. Metascape analysis (Zhou et al. 2019) was performed with upregulated genes of a log 2 -fold change cut-off of 2.0 or greater and ensembl ID lists were uploaded respectively per comparison condition. Statistical significance was calculated utilizing three biological replicates for each exposure condition and genotype, and was determined using student's unpaired t-test.

APAP can be bioactivated by a cytochrome P450 enzyme, Cyp2e1, to catalyze the formation of the reactive metabolite NAPQI (Fig. 1A) . The response to toxic levels of APAP (600 mg/kg) was studied in 8-12-week-old C57BL/6 male WT and Alkbh8 Def mice. Stress biomarkers indicative of liver damage and oxidative stress were measured in serum derived from whole blood and liver tissue, respectively. Alanine aminotransferase (ALT) levels were significantly higher in Alkbh8 Def livers 6 hr after injection of APAP, relative to APAP exposed WT livers (Fig. 1B) . The lipid peroxidation product 8-isoprostane is an in vivo biomarker of oxidative stress (Montuschi, Barnes, and Roberts 2004) . For both saline and 6 hr APAP exposure conditions for Alkbh8 Def liver tissue there was a slight increase in 8-isoprostane levels compared to WT samples (Fig.1C) , but it did not reach a significant level (p = 0.33). Glutathione peroxidase 3 (Gpx3) is an extracellular antioxidant enzyme that aids in scavenging hydrogen peroxide as well as other hydroperoxides (Takebe et al. 2002) . 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 Alkbh8 Def liver tissue (Fig.1D) .

Daily injections over 4 days were used to study chronic APAP use in the WT and Alkbh8 Def mice. ALT (p = 0.004) and 8-isprostane levels (p = 0.0001) were measured and found to be significantly increased in the Alkbh8 Def mice after APAP injection compared to WT APAP ( Fig. 1E-F) . Gpx3 expression levels in WT and Alkbh8 Def livers were also significantly (p = 0.02) different after 4 days of APAP exposure (Fig. 1G ). Similar to 6-hour data, there was an APAPinduced increase in Gpx3 in WT livers, which was not observed in the Alkbh8 Def mice. Our ALT, 8-isoprostane and Gpx3 data support the idea that writer deficient livers are experiencing significant APAP induced stress and defective selenoprotein synthesis.

We next analyzed gene expression using mRNA-seq of livers from saline and APAP- binding (Strausberg et al. 2002 ) and a member of the MYC Proto-Oncogene (myc) family that is highly expressed in the brain (Bmyc) and functions as a transcriptional regulator. We also report similar gene transcript profiles when comparing WT and Alkbh8 Def under saline control conditions (Fig. S1 ). The similarities in the transcriptional responses to APAP between the two genotypes was expected, as Alkbh8, along with its accessory protein Trm112, post transcriptionally regulate gene expression by modifying tRNA Sec (Fig. 3B-D) and other tRNAs.

Alkbh8 can modify tRNA Sec , which is used in stop codon recoding, to drive the translation of 25 selenocysteine containing proteins in the Mus musculus genome (Fig. 3D) . Other regulatory components tied to stop codon recoding include the cis Sec insertion sequence (SECIS), a stem-loop structure located in coding regions and in 3'UTRs of eukaryotic genes (Low & Berry, 1996; Berry, J et al., 1991) .

We next analyzed for differences in post transcriptional regulation by quantifying where we measured a significant (p = 0.01) increase from 0.004 pg/µL to 0.045 pg/µL in saline and APAP conditions, respectively. The failure to increase epitranscriptomic marks in tRNA Sec in response to APAP in Alkbh8 Def liver tissue predicts that selenoproteins levels would be affected. We quantified Gpx1 and Trxr2 levels (Fig. 4C ) and similar to Gpx3 (Fig. 1D) , Gpx1

and Trxr2 were increased in WT livers exposed to APAP with Trxr2 being significant (p = 0.002);

this stress induced increase in protein levels was absent in Alkbh8 Def livers. Gpx4, Trxr1 and selenoprotein S (SelS) also failed to respond to APAP treatment in Alkbh8 Def animals (Supplemental Fig. S4 ).

We measured 37 tRNA modifications using LC-MS/MS and compared their levels, relative to WT saline, using a heat map (Fig. 5A-B) . The WT response to APAP resulted in 31 total increased modifications with 22 of the group being significant ( (Fig. 5B) . Other modifications are shown in (Supplemental Fig. S5) . The tRNA modifications that were the most downregulated in the Alkbh8 Def APAP samples were; T (-6.5 log 2 fold change, p < 0.05), mnm 5 U (-6.4 log 2 fold change, p < 0.05) and mcm 5 U (-3.7 log 2 fold change, p < 0.05). Our epitranscriptomic data provides strong support for the idea that a single acute toxic dose of APAP promotes significant changes in many tRNA modifications. Below we explore whether chronic APAP exposure promote changes to the epitranscriptome.

We analyzed RNA from mice on the 4 th day of daily APAP treatments. mRNA sequencing experiments revealed many (N=3) differences in gene regulation between WT and

Alkbh8 Def in the response to APAP toxicity at the 4 th day. The comparison of mRNA-seq data from WT APAP vs. WT saline identified significantly upregulated genes (p ≤ 0.05) in pathways related to response to stillbenoid, inflammatory response and unsaturated fatty acid metabolic process (Fig. 6B) . protein levels were increased in WT APAP samples in response to saline, which was not observed in Alkbh8 Def livers (Fig. 6D) . Alkbh8 Def liver tissue compared to WT after daily APAP exposure including: m 5 U, mcm 5 s 2 U, mnm 5 U, mo 5 U, mnm 5 s 2 U, and s 4 U. It is interesting to note that both s 2 U and s 2 mo 5 U concentration were measured to be increased after daily APAP exposure in Alkbh8 Def mice compared to WT. Adenosine-based modifications m 1 A, and m 6 A were also measured to be dysregulated in the Alkbh8 Def liver tissue compared to WT after daily APAP exposure. All measured modifications within the chronic timeline are shown in (Supplemental Fig. 7) and

(Supplemental Table 1 ). . Wobble U writers play important translational regulatory roles that protect normal cells and organs from stress, and our studies highlight that the wobble U writer Alkbh8 helps regulate the response to APAP toxicity.

Protein kinase cascades are well described responses to APAP, as it activates the c-Jun-N-terminal kinase (JNK) through the sequential protein phosphorylation of mainly mitogenactivated protein kinases (MAPK) (Harrison et al. 2004; Johnson and Lapadat 2002) . JNK is a critical member of the MAPK family and considered a control point between physiological and pathological status. The activation of JNK has shown to be important in biological processes including stress reaction, cellular differentiation, and apoptosis (Harrison et al. 2004; Johnson and Lapadat 2002) . The activated JNK can translocate to the mitochondria and binds to an outer membrane protein known as Sab, which is also phosphorylated. JNK binding to Sab leads to further ROS generation and sustains the activation of JNK leading to the induction of the mitochondrial permeability transition (MPT) resulting in the mediation of hepatocyte necrosis (Win et al. 2011) . c-Myc is induced by APAP and encompasses one of the most significant protein networks that are associated with liver injury (Beyer et al. 2007 ). Beyer et al., (Beyer et al. 2007 ) presented gene ontology analysis of transcripts that significantly correlate with liver necrosis and the resulting biological pathways 6 hours post-APAP exposure included programmed cell death, response to wounding, and inflammatory response. These biological pathways are upregulated in both genotypes of mice after 6 hr APAP exposure when compared to their corresponding saline control. Our transcriptional studies support the idea that both WT and Alkbh8 Def mice respond to APAP toxicity similarly at the mRNA level.

Our study is unique in that we describe a change in RNA modification regulated protein expression in response to APAP stress. Members of the Gpx and TrxR selenoprotein families are known to be critical for signaling and aiding in protection and reduction of harmful oxidants ). Gpx and TrxR selenoproteins have also been shown to be key regulators of mitochondrial oxidative stress. Inhibition of these antioxidant enzymes with Auranofin in noncancerous human Swan-71 cells resulted in reduction of cell viability, mitochondrial respiration, and increased oxidative stress. Selenium supplementation was able to restore Gpx and TrxR selenoprotein activity which subsequently decreased ROS production and increased cell viability (Radenkovic et al. 2017) . These and other studies (Lopert, Day, and Patel 2012; Miyamoto et al. 2003) confirm the important role of Gpx and TrxR selenoproteins as key response regulators to mitochondrial oxidant stress. Furthermore, APAP hepatoxicity has been reported to result in the formation of the potent oxidant peroxynitrite from superoxide and nitric oxide (Hinson et al. 1998) which was subsequently observed to be predominately generated inside the mitochondria (Cover et al. 2005 studies in cancer models support the idea that this regulation will be extended to many types of protein families and include regulation of the 20 standard amino acids (Rapino et al. 2017 (Rapino et al. , 2018 .

tRNA modifications have been shown to be a critical part of epitranscriptomic reprogramming in response to stressors, chemicals and physiological states that have been shown in many organisms. (Chan et al. 2012; Endres, Dedon, and Begley 2015; Gu, Begley, and Dedon 2014) . Our study highlights that APAP promotes a global reprogramming of RNA modifications. The tRNA Sec modification, i 6 A was elevated in WT in response to APAP however this response was again disrupted in the Alkbh8 Def mice. TRIT1 is responsible for modifying position 37 in the anticodon loop of tRNA Sec . The i 6 A modification can also be found on various cytoplasmic or mitochondrial tRNA. Cytoplasmic i 6 A modification occurs on tRNA Ser and mitochondrial tRNAs, mtRNA Cys and mtRNA Ser (Schweizer, Bohleber, and Fradejas-Villar 2017) .

A reduced expression of Trit1 may account for decreased selenoprotein expression (Warner et al. 2000) . Interestingly, an A37G mutation that abolishes the i 6 A37 modification supports a subset of selenoprotein expression including Txnrd1 and Gpx4; however, there was little to no effect on a subset of stress-related selenoproteins such as Gpx1 (Warner et al. 2000) . The increase in mcm 5 U and tRNA Sec related modifications suggest that the WT livers respond to APAP by optimizing the production of selenoproteins.

A comparison of saline derived RNA modification data between WT and Alkbh8 Def , revealed that the tRNA modification m 1 A was found at similar levels. After APAP exposure, WT responds by increasing m 1 A modification levels and this reprogramming is disrupted in the Alkbh8 Def mice. m 1 A is present in both cytosolic tRNA species at positions 9, 14, 22,5 7 and 58, and notably on tRNA Sec at position 58, as well as mitochondrial tRNA species at positions 9 and 58 (Ali, Idaghdour, and Hodgkinson 2020; Safra et al. 2017 ). The addition of the m 1 A modification is catalyzed by the catalytic protein Trmt61A and the RNA binding protein Trmt6 (M. Wang et al. 2016 ) while the eraser enzyme nucleic acid dioxygenase (Alkbh1) demethylates the m 1 A modification reverting to its unmodified form. Alkbh1 impacts tRNA stability and regulates cleavage dependent on the particular stress response (Rashad et al. 2020) . The m 1 A modification aids in mitochondrial tRNA folding and ensures correct structure formation. The m 1 A58 tRNAs have a differential affinity for the elongation factor EF1A which delivers the tRNA into the A-site of the ribosome in protein synthesis (Pan 2018) . Our data supports the idea that m 1 A plays an important translational role in protein synthesis in response to APAP and the deficiency of Alkbh8 dysregulates the epitranscriptomic reprogramming. Likely the m 1 A deficiency contributes to the decrease in selenoprotein synthesis, but its decrease could also be globally affecting protein synthesis in the cytoplasm and mitochondria. In addition the m 1 A decrease could be promoting tRNA instability, which could account for the wide-spread decrease in RNA modifications in APAP treated Alkbh8 Def livers.

The m 3 C modification was significantly up-regulated in WT livers after APAP exposure.

The m 3 C modification is located at position 32 on various tRNA species as well as their corresponding isoacceptors including; tRNA Arg , tRNA Met , tRNA Ser , tRNA Thr and mt-tRNA Met , mt-tRNA Ser , mt-tRNA Thr ). The m 3 C modification is catalyzed by the methyltransferase-like protein 6 (METTL6) (Xu et al. 2017 ) and human METTL6 has been associated with regulating cellular growth, ribosomal occupancy, and pluripotency in hepatocellular carcinoma (HCC). Mettl6 knockout mice displayed altered metabolic activity via altered glucose homeostasis, changes in metabolic turnover, and decreased liver weight suggesting the impact Mettl6 has on hepatic growth (Ignatova et al. 2020 ). to activate a dioxygene molecule (Fedeles et al. 2015) . The dioxygenases use oxygen as an electron acceptor to reduce methylation and to form formaldehyde and hydrogen peroxide which may suppress the overproduction of oxidative stress (Chervona and Costa 2012) . It is suggested that oxidative stress oxidizes Fe (II) to Fe (III) inhibiting the demethylase activity of eraser enzymes such as FTO (Niu et al. 2015; Ponnaluri, Maciejewski, and Mukherji 2013) .

However, overexpression of FTO in hepatocytes and myotubes has been linked to increasing lipogenesis and mitochondrial dysfunction leading to increased oxidative stress (Bravard et al. 2011; Guo et al. 2013) . Reader enzymes YTHDF1-3 studies have also been shown to have various roles in recognizing oxidative stress signaling and promote regulation of antioxidant pathways, in addition to influencing mRNA stability and translation (Anders et al. 2018; Shi et al. 2019) . m 5 C writer enzymes have been shown in HeLa and colon cancer cell lines to upregulate NRF2 and induce cellular senescence upon oxidative stress exposure (Q. Li et al. 2018; Villeneuve et al. 2009 ). Together with our current Alkbh8 study, past studies highlight the importance of epitranscriptomic marks in response to stress and link mcm 5 U, m 1 A, m 3 C, m 6 A and m 5 C key to the response to ROS.

Our study highlights that APAP promotes global reprogramming of the epitranscriptome and its effects can be in part modulated by a single epitranscriptome writer (Fig. 7) . The deficiency of Alkbh8 resulted in increased liver damage and oxidative stress in Alkbh8 Def mice upon APAP exposure, which can be linked to a global disruption of epitranscriptomic reprogramming, a specific decrease in wobble U modification and the decreased translation of stress response proteins. Chronic exposure of APAP reveals further dysregulation of these critical epitranscriptomic reprogramming efforts, together with global mRNA changes, likely resulting from mitochondrial stress, which leads to additional DNA damage on top of the other pathological consequences. A major finding of our study is that pharmaceuticals promote changes to the epitranscriptome and RNA modification enzymes can modulate outcomes. The efficacy of chemotherapeutics and immunotherapies have also been shown to be modulated by epitranscriptomic systems. METTL3 expression increases resistance to the chemotherapeutic agents 5-fluorouracil, gemcitabine, and cisplatin in pancreatic cancer, through activation of the MAPK signaling pathway (Taketo et al. 2018) . The eraser enzyme, FTO, promotes melanoma tumorigenesis and resistance to immunotherapy, while decreased FTO expression increased

IFNy-induced tumor cell killing and promoted beneficial PD-1 immunotherapy (Yang et al. 2019) .

Similarly, decreased expression of the eraser ALKBH5 increased the efficiency of PD-1 therapy in both melanoma and colorectal cancer models by decreasing immunosuppressive cells (N. Li et al. 2020) . Additionally, wobble U34 modifications catalyzed by ELP3/CTU1/2 have been shown to drive melanomas that express mutated BRAF, with subsequent resistance to anti-BRAF therapy caused by U34-dependent codon-biased translation of metabolic proteins (Rapino et al. 2017) . It is important to note that many of the previously published studies

showing that the efficacy of therapeutics can be modulated by epitranscriptomic marks were done using in situ models and focus on agents specific to cancer treatment. Our study presents an in vivo model of how a writer can modulate pharmaceutical stress resulting from a pain reliever and suggests that epitranscriptomic modulation could augment other therapeutics.

Overexpression of Alkbh8 or increased wobble modifications in tRNA Sec could limit APAP toxicity and provide an increased protective role against oxidative stress. Notably, selenium supplementation has been shown to promote increased wobble modification of tRNA Sec and promote selenoprotein expression (Chittum et al. 1997) , and it could offer an effective epitranscriptomic enhancement strategy to counteract APAP overdose. Calculations for each epitranscriptomic mark and comparisons between WT and Alkbh8 Def liver tissue post 6-hour exposure to 600 mg/kg of APAP with reported statistical significance of biological replicates (N = 3) measured by an unpaired t-test. WT and Alkbh8 Def mice (N = 3) were left untreated or exposed to a single dose of 600 mg/kg APAP and livers were harvested after 6 hours and RNA was purified and subject to mRNA-seq. Selenoprotein levels were evaluated using the ProteinSimple WES system. Statistical significance of biological replicates (N = 3) was determined using an unpaired t-test with ( * p < 0.05, **p < 0.01, *** p < 0.001). Supplemental Tables   Table S1 . Measured tRNA modifications in mouse liver tissue after daily dose of APAP (4

Alkbh8 Def liver tissue post daily 4 Day exposure to 600 mg/kg of APAP. Table S2 . WES raw data for all proteins analyzed in 6 hour APAP exposure experiment.

Protein quantitation data was normalized to housekeeping protein, GAPDH, and normalized corrected area analysis setting was set to 100 on ProteinSimple Compass Software. Table S3 . WES raw data for all proteins analyzed in 4 day APAP exposure experiment.

Protein quantitation data was normalized to housekeeping protein, GAPDH, and normalized corrected area analysis setting was set to 100 on ProteinSimple Compass Software. protein levels in the liver were evaluated using the ProteinSimple WES system. Statistical significance of biological replicates (N = 3) was measured by an unpaired t-test with ( * p < 0.05, **p < 0.01, *** p < 0.001). Figure S5 . Remainder of measured epitranscriptomic marks in mouse liver tissue after APAP (6 hours). WT and Alkbh8 Def mice (N = 3) were exposed to a single 600 mg/kg dose of APAP and livers were harvested 6 hours after dosing. Modifications were measured using LC-MS/MS. Statistical significance of biological replicates (N = 3) was measured by an unpaired ttest with ( * p < 0.05, **p < 0.01, *** p < 0.001).

Def liver tissue after 4 day APAP exposure. SelS expression was evaluated using the ProteinSimple WES system. Statistical significance of biological replicates (N = 3) was determined using an unpaired t-test with ( * p < 0.05, **p < 0.01, *** p < 0.001). 

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We thank Dr. Sridar Chittur and Marcy Kuentzel from the Center for Functional Genomics at the University at Albany for their help and collaboration with our RNASeq experiments. We also thank Dr. Kevin O'Keefe for his expertise with mouse work along with Ed Zandro M. Taroc and his help with bioinformatic analysis. We finally thank members of the University at Albany RNA Institute and the Nanobioscience constellation at SUNY Poly's College of Nanoscale College and Engineering for their insightful discussion and feedback.