N⁶-methyladenosine (M⁶A) in fetal offspring modifies mitochondrial gene expression following gestational nano-TiO₂ inhalation exposure

Abstract

N⁶-methyladenosine (m⁶A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it’s role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO₂) contributes to cardiac m⁶A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO₂ with a target aerosol mass concentration of 12 mg/m³. At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m⁶A antibodies was performed followed by sequencing of immunoprecipitant (m⁶A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO₂ exposed mice (Padj ≤ 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m⁶A methylation sites (Padj ≤ 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P ≤ 0.05). The lack of m⁶A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO₂ inhalation exposure.

Keywords:

• Epitranscriptomics
• heart
• 3’ uTR
• m⁶A
• proteomics
• Multi-Omics

Acknowledgments

We would like to acknowledge the WVU Genomics Core Facility, Morgantown WV for support provided to help make this publication possible and CTSI Grant #U54 GM104942 which in turn provides financial support to the Core Facility. We would like to thank the Center for Inhalation Toxicology (iTOX) at West Virginia University for access to the facility and coordination of timed exposures. We would like to thank Sherri A. Friend and the National Institute for Occupational Safety and Health, Morgantown, WV, USA for contributing to the physicochemical characterization of the nano-TiO₂ aerosolized particles.

Ethical approval and consent to participate

The West Virginia University Animal Care and Use Committee approved all animal studies, which conformed to the most current National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals manual.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All code used in the bioinformatic processing of samples is provided in the GitHub repository, https://github.com/qahathaway/mRNA-m6A-Proteomics. All data generated or analyzed during this study are included in this published article and its supplementary information files, as well as on the GitHub repository. Raw label-free proteomics data available upon request. Raw sequencing files have been uploaded to Gene Expression Omnibus (GEO) under accessions:

1. GSE211479 – An Adaptive Response through N6-Methyladenosine in Fetal Offspring following Gestational Nano-TiO2 Inhalation Exposure I

2. GSE211480 – An Adaptive Response through N6-Methyladenosine in Fetal Offspring following Gestational Nano-TiO2 Inhalation Exposure II

3. GSE211481 – An Adaptive Response through N6-Methyladenosine in Fetal Offspring following Gestational Nano-TiO2 Inhalation Exposure

Additional information

Funding

This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) under grants [R01 HL128485] (JMH) and [R01 HL168290] (JMH), the National Institute of Environmental Health Sciences (NIEHS) under grant [R01 ES034628] (JMH), the American Heart Association (AHA) under grant [AHA-20PRE35080170] (AK), grant [AHA-17PRE33660333] (QAH), and grant [AHA-829079] (ADT), and the Community Foundation for the Ohio Valley Whipkey Trust (JMH).