Hypoxia-induced downregulation of RNA m6A protein machinery in the naked mole-rat heart.

Naked mole-rats, Heterocephalus glaber, are champion hypoxia-tolerant rodents that live under low oxygen conditions in their subterranean burrows. Detrimental effects of low oxygen can be mitigated through metabolic rate depression (MRD), metabolic reorganization, and global downregulation of nonessential cellular processes. Recent research has progressively implicated epigenetic modifications - rapid, reversible changes to gene expression that do not alter the DNA sequence itself - as major players in implementing and maintaining MRD. N6-adenosine (m6A) methylation is the most prevalent mammalian RNA modification and is responsible for pre-mRNA processing and mRNA export from the nucleus. Hence, m6A -mediated conformational changes alter the cellular fate of transcripts. The present study investigated the role of m6A RNA methylation responses to 24 h of hypoxia exposure in H. glaber cardiac tissue. Total protein levels of m6A writers/readers/erasers, m6A demethylase activity, and total m6A quantification were measured under normoxic vs. hypoxic conditions in H. glaber heart. While there was no change in either demethylase activity or total m6A content, many proteins of the m6A pathway were downregulated during hypoxia. Overall, m6A may not be a signature hypoxia-responsive characteristic in H. glaber heart, but downregulation of the protein machinery involved in m6A cycling points to an alternate biological involvement. Further research will explore other forms of RNA modifications and other epigenetic mechanisms to determine the controls on hypoxia endurance in this subterranean mammal.

Hibernation-Induced microRNA Expression Promotes Signaling Pathways and Cell Cycle Dysregulation in Ictidomys tridecemlineatus Cardiac Tissue.

The thirteen-lined ground squirrel Ictidomys tridecemlineatus is a rodent that lives throughout the United States and Canada and uses metabolic rate depression to facilitate circannual hibernation which helps it survive the winter. Metabolic rate depression is the reorganization of cellular physiology and molecular biology to facilitate a global downregulation of nonessential genes and processes, which conserves endogenous fuel resources and prevents the buildup of waste byproducts. Facilitating metabolic rate depression requires a complex interplay of regulatory approaches, including post-transcriptional modes such as microRNA. MicroRNA are short, single-stranded RNA species that bind to mRNA transcripts and target them for degradation or translational suppression. Using next-generation sequencing, we analyzed euthermic vs. hibernating cardiac tissue in I. tridecemlineatus to predict seven miRNAs (let-7e-5p, miR-122-5p, miR-2355-3p, miR-6715b-3p, miR-378i, miR-9851-3p, and miR-454-3p) that may be differentially regulated during hibernation. Gene ontology and KEGG pathway analysis suggested that these miRNAs cause a strong activation of ErbB2 signaling which causes downstream effects, including the activation of MAPK and PI3K/Akt signaling and concurrent decreases in p53 signaling and cell cycle-related processes. Taken together, these results predict critical miRNAs that may change during hibernation in the hearts of I. tridecemlineatus and identify key signaling pathways that warrant further study in this species.

MicroRNA transcriptomics in liver of the freeze-tolerant gray tree frog (Dryophytes versicolor) indicates suppression of energy-expensive pathways.

The rapid and reversible nature of microRNA (miRNA) transcriptional regulation is ideal for implementing global changes to cellular processes and metabolism, a necessary asset for the freeze-tolerant gray tree frog (Dryophytes versicolor). D. versicolor can freeze up to 42% of its total body water during the winter and then thaw completely upon more favorable conditions of spring. Herein, we examined the freeze-specific miRNA responses in the gray tree frog using RBiomirGS, a bioinformatic tool designed for the analysis of miRNA-seq transcriptomics in non-genome sequenced organisms. We identified 11 miRNAs differentially regulated during freezing (miR-140-3p, miR-181a-5p, miR-206-3p, miR-451a, miR-19a-3p, miR-101-3p, miR-30e-5p, miR-142-3p and -5p, miR-21-5p, and miR-34a-5p). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggests these miRNAs play roles in downregulating signaling pathways, apoptosis, and nuclear processes while enhancing ribosomal biogenesis. Overall, these findings point towards miRNA inducing a state of energy conservation by downregulating energy-expensive pathways, while ribosomal biogenesis may lead to prioritization of critical processes for freeze-tolerance survival.

Small RNA sequencing in hypoxic naked mole-rat hearts suggests microRNA regulation of RNA- and translation-related processes.

The naked mole-rat (Heterocephalus glaber) regularly endures intermittent periods of hypoxia in its burrows, surviving in part due to metabolic rate depression (MRD)-a strategy of conserving cellular resources by downregulating nonessential gene expression and reorganizing cellular processes. miRNA are short, noncoding RNAs already implicated for their roles in numerous models of extreme environmental stress; given their rapid, reversible nature, they are ideal for implementing MRD. We performed small RNA sequencing on cardiac tissue from normoxic versus 24 h hypoxic naked mole-rats, and used bioinformatics to predict 18 miRNAs which may be differentially regulated during hypoxia. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway mapping further suggest these miRNAs play roles in largely translation-related functions, including RNA processing and catabolism.

MicroRNA biogenesis proteins follow tissue-dependent expression during freezing in Dryophytes versicolor.

Grey tree frogs (Dryophytes versicolor) have the remarkable ability to endure full-body freezing over the winter, with up to 42% of total body water converted into extracellular ice. Survival is aided by metabolic rate depression that greatly reduces tissue energy costs over the winter. Post-transcriptional controls on gene expression which include miRNA regulation of gene transcripts can aid implementation of the reversible changes required for freeze tolerance, since miRNAs are ideal for facilitating the rapid metabolic reorganization needed for this process. The energy cost for synthesizing new miRNAs is low, and miRNAs' ability to target more than one mRNA transcript (and vice versa) allows a wide versatility in their capability for metabolic restructuring. Western immunoblotting was used to examine protein expression levels of members of the miRNA biogenesis pathway in D. versicolor liver, skeletal muscle, and kidney. Four of these proteins (Dicer, Drosha, Trbp, Xpo5) were upregulated in liver of frozen frogs, suggesting enhanced capacity for miRNA biogenesis, whereas expression of four proteins in frozen muscle (Ago1, Ago2, Dgcr8, Xpo5) and six proteins in kidney (Ago1, Ago2, Ago3, Ago4, Dgcr8, Ran-GTP) were downregulated, indicating an opposite trend. Overall, the data show that miRNA biosynthesis is altered during freezing and differentially regulated across tissues. We suggest that miRNAs are central for the freeze tolerance strategy developed by D. versicolor, and future research will expound upon specific miRNAs and their roles in mediating responses to freezing stress.

Pro- and anti-apoptotic microRNAs are differentially regulated during estivation in Xenopus laevis.

Xenopus laevis, the African clawed frog, undergoes seasonal estivation to survive periods of drought when its lake-bed habitats dry up. The frog can lose ∼30% of its total body water, leading to conditions of impaired blood flow and ischemia which risk cellular survival under these harsh conditions. MicroRNAs are short, noncoding, single-stranded RNAs 21-24 nt long that have been widely implicated in hypometabolic responses, and serve functions including apoptosis survival. The levels of three pro-apoptotic and four anti-apoptotic miRNAs were measured in liver and skeletal muscle of estivating X. laevis, and bioinformatic analysis was performed to verify potential mRNA targets of these miRNAs. Members of pro-apoptotic miRNAs miR-15a, miR-16, and miR-101 showed upregulation as a result of dehydration stress, while anti-apoptotic miRNAs miR-19b, miR-21, miR-92a, and miR-155 showed differential regulation between the two tissues. Together, these miRNAs act in a more diverse fashion than arbitrarily pro- or anti-apoptotic, and encompass functions ranging from the inhibition of cell proliferation through cell cycle arrest to the prevention of skeletal muscle atrophy.

Muscles in Winter: The Epigenetics of Metabolic Arrest.

The winter months are challenging for many animal species, which often enter a state of dormancy or hypometabolism to "wait out" the cold weather, food scarcity, reduced daylight, and restricted mobility that can characterize the season. To survive, many species use metabolic rate depression (MRD) to suppress nonessential metabolic processes, conserving energy and limiting tissue atrophy particularly of skeletal and cardiac muscles. Mammalian hibernation is the best recognized example of winter MRD, but some turtle species spend the winter unable to breathe air and use MRD to survive with little or no oxygen (hypoxia/anoxia), and various frogs endure the freezing of about two-thirds of their total body water as extracellular ice. These winter survival strategies are highly effective, but create physiological and metabolic challenges that require specific biochemical adaptive strategies. Gene-related processes as well as epigenetic processes can lower the risk of atrophy during prolonged inactivity and limited nutrient stores, and DNA modifications, mRNA storage, and microRNA action are enacted to maintain and preserve muscle. This review article focuses on epigenetic controls on muscle metabolism that regulate MRD to avoid muscle atrophy and support winter survival in model species of hibernating mammals, anoxia-tolerant turtles and freeze-tolerant frogs. Such research may lead to human applications including muscle-wasting disorders such as sarcopenia, or other conditions of limited mobility.

Epigenetic underpinnings of freeze avoidance in the goldenrod gall moth, Epiblema scudderiana.

The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero temperatures during the winter by supercooling bodily fluids to approximately -40 °C, allowing the insect to remain unfrozen despite the freezing temperatures. This is characterized by a drastic increase of cryoprotectant glycerol along with widespread downregulation of non-essential genes and processes to conserve cellular energy. This study examined the role of epigenetic enzymes in regulating this freeze-avoidant process across a range of freezing temperatures experienced in nature. Cold and subzero temperature exposure in E. scudderiana resulted in upregulation of select DNA methyltransferase (DNMT) enzymes with concurrent decreases in DNMT activity and no change in activity of the Ten-Eleven Translocation (TET) demethylation enzyme activities. Levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity decreased during cold exposures. The increase in DNMT expression and concurrent decrease in HAT activity suggests a role for DNA methylation to assist with transcriptional suppression. These findings propose that epigenetic regulation of genes and histones underpin the winter survival strategies of this insect.

Epigenetic regulation by DNA methyltransferases during torpor in the thirteen-lined ground squirrel Ictidomys tridecemlineatus.

The thirteen-lined ground squirrel, Ictidomys tridecemlineatus, is a mammal capable of lowering its Tb to almost 0 °C while undergoing deep torpor bouts over the winter. To decrease its metabolic rate to such a drastic extent, the squirrel must undergo multiple physiological, biological, and molecular alterations including downregulation of almost all nonessential processes. Epigenetic regulation allows for a dynamic range of transient phenotypes, allowing the squirrel to downregulate energy-expensive and nonessential pathways during torpor. DNA methylation is a prominent form of epigenetic regulation; therefore, the DNA methyltransferase (DNMT) family of enzymes were studied by measuring expression and activity levels of the five major proteins during torpor bouts. Additionally, specific cytosine marks on genomic DNA were quantified to further elucidate DNA methylation during hibernation. A tissue-specific response was observed that highlighted variant degrees of DNA methylation and DNMT expression/activity, demonstrating that DNA methylation is a highly complex form of epigenetic regulation and likely one of many regulatory mechanisms that enables metabolic rate depression in response to torpor.