Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation.

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.

c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

Adrenal gene expression dynamics support hibernation in 13-lined ground squirrels.

Hibernation is a natural model of extreme physiology in a mammal. Throughout winter, small hibernators repeatedly undergo rapid, dramatic swings in body temperature, perfusion, and oxygen delivery. To gain insight into the molecular mechanisms that support homeostasis despite the numerous challenges posed by this dynamic physiology, we collected 13-lined ground squirrel adrenal glands from at least five individuals representing six key timepoints across the year using body temperature telemetry. Differentially expressed genes were identified using RNA-seq, revealing both strong seasonal and torpor-arousal cycle effects on gene expression. Two novel findings emerge from this study. First, transcripts encoding multiple genes involved in steroidogenesis decreased seasonally. Taken together with morphometric analyses, the data are consistent with preservation of mineralocorticoids but suppression of glucocorticoid and androgen output throughout winter hibernation. Second, a temporally orchestrated, serial gene expression program unfolds across the brief arousal periods. This program initiates during early rewarming with the transient activation of a set of immediate early response (IER) genes, comprised of both transcription factors and the RNA degradation proteins that assure their rapid turnover. This pulse in turn activates a cellular stress response program to restore proteostasis comprised of protein turnover, synthesis, and folding machinery. These and other data support a general model for gene expression across the torpor-arousal cycle that is facilitated in synchrony with whole body temperature shifts; induction of the immediate early response upon rewarming activates a proteostasis program followed by a restored tissue-specific gene expression profile enabling renewal, repair, and survival of the torpid state.NEW & NOTEWORTHY This pioneer study of adrenal gland gene expression dynamics in hibernating ground squirrels leverages the power of RNA-seq on multiple precisely timed samples to demonstrate: 1) steroidogenesis is seasonally reorganized to preserve aldosterone at the expense of glucocorticoids and androgens throughout winter hibernation; 2) a serial gene expression program unfolds during each short arousal whereby immediate early response genes induce the gene expression machinery that restores proteostasis and the cell-specific expression profile before torpor reentry.

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.

Dynamic temperature-sensitive A-to-I RNA editing in the brain of a heterothermic mammal during hibernation.

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including Drosophila and Cephalopoda, where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator.

A functional transcriptomic analysis in the relict marsupial Dromiciops gliroides reveals adaptive regulation of protective functions during hibernation.

The small South American marsupial, Dromiciops gliroides, known as the missing link between the American and the Australian marsupials, is one of the few South American mammals known to hibernate. Expressing both daily torpor and seasonal hibernation, this species may provide crucial information about the mechanisms and the evolutionary origins of marsupial hibernation. Here, we compared torpid and active individuals, applying high-throughput sequencing technologies (RNA-seq) to profile gene expression in three D. gliroides tissues and determine whether hibernation induces tissue-specific differential gene expression. We found 566 transcripts that were significantly up-regulated during hibernation (369 in brain, 147 in liver and 50 in skeletal muscle) and 339 that were down-regulated (225 in brain, 79 in liver and 35 in muscle). The proteins encoded by these differentially expressed genes orchestrate multiple metabolic changes during hibernation, such as inhibition of angiogenesis, prevention of muscle disuse atrophy, fuel switch from carbohydrate to lipid metabolism, protection against reactive oxygen species and repair of damaged DNA. According to the global enrichment analysis, brain cells seem to differentially regulate a complex array of biological functions (e.g., cold sensitivity, circadian perception, stress response), whereas liver and muscle cells prioritize fuel switch and heat production for rewarming. Interestingly, transcripts of thioredoxin-interacting protein (TXNIP), a potent antioxidant, were significantly over-expressed during torpor in all three tissues. These results suggest that marsupial hibernation is a controlled process where selected metabolic pathways show adaptive modulation that can help to maintain homeostasis and enhance cytoprotection in the hypometabolic state.

Characterization of miRNAs modulated by torpor in the hibernating ground squirrel Ictidomys tridecemlineatus liver by next-generation sequencing.

BACKGROUND: Mammalian hibernation is a fascinating phenomenon that involves multiple molecular and biochemical changes to proceed. While the molecular picture associated with torpor has become clearer in recent years, the function of non-coding RNAs, and especially of microRNAs, solicited during this process is not well understood. OBJECTIVE: To better characterize a signature of cold torpor-associated miRNAs in the hibernating thirteen-lined ground squirrel Ictidomys tridecemlineatus. MATERIALS AND METHODS: Next-generation sequencing and qRT-PCR approaches were conducted in euthermic and hibernating ground squirrel liver tissues. RESULTS: This high-throughput approach notably revealed modulation during hibernation of various miRNAs previously associated with lipid metabolism, glucose metabolism and antioxidant responses such as miR-145a-3p, miR-22-3p and miR-25-3p, respectively. CONCLUSION: Overall, these results present a group of miRNAs differentially expressed in hibernating ground squirrel liver and provide additional knowledge on the underlying functions of these small non-coding molecules during cold torpor.

Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels.

Mammalian hibernators adapt to prolonged periods of immobility, hypometabolism, hypothermia, and oxidative stress, each capable of reducing bone marrow activity. In this study bone marrow transcriptomes were compared among thirteen-lined ground squirrels collected in July, winter torpor, and winter interbout arousal (IBA). The results were consistent with a suppression of acquired immune responses, and a shift to innate immune responses during hibernation through higher complement expression. Consistent with the increase in adipocytes found in bone marrow of hibernators, expression of genes associated with white adipose tissue are higher during hibernation. Genes that should strengthen the bone by increasing extracellular matrix were higher during hibernation, especially the collagen genes. Finally, expression of heat shock proteins were lower, and cold-response genes were higher, during hibernation. No differential expression of hematopoietic genes involved in erythrocyte or megakaryocyte production was observed. This global view of the changes in the bone marrow transcriptome over both short term (torpor vs. IBA) and long term (torpor vs. July) hypothermia can explain several observations made about circulating blood cells and the structure and strength of the bone during hibernation.

Analysis of microRNA expression during the torpor-arousal cycle of a mammalian hibernator, the 13-lined ground squirrel.

Hibernation is a highly regulated stress response that is utilized by some mammals to survive harsh winter conditions and involves a complex metabolic reprogramming at the cellular level to maintain tissue protections at low temperature. In this study, we profiled the expression of 117 conserved microRNAs in the heart, muscle, and liver of the 13-lined ground squirrel (Ictidomys tridecemlineatus) across four stages of the torpor-arousal cycle (euthermia, early torpor, late torpor, and interbout arousal) by real-time PCR. We found significant differential regulation of numerous microRNAs that were both tissue specific and torpor stage specific. Among the most significant regulated microRNAs was miR-208b, a positive regulator of muscle development that was found to be upregulated by fivefold in the heart during late torpor (13-fold during arousal), while decreased by 3.7-fold in the skeletal muscle, implicating a potential regulatory role in the development of cardiac hypertrophy and skeletal muscle atrophy in the ground squirrels during torpor. In addition, the insulin resistance marker miR-181a was upregulated by 5.7-fold in the liver during early torpor, which supports previous suggestions of hyperinsulinemia in hibernators during the early stages of the hibernation cycle. Although microRNA expression profiles were largely unique between the three tissues, GO annotation analysis revealed that the putative targets of upregulated microRNAs tend to enrich toward suppression of progrowth-related processes in all three tissues. These findings implicate microRNAs in the regulation of both tissue-specific processes and general suppression of cell growth during hibernation.

Thyroid hormone status affects expression of daily torpor and gene transcription in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones (TH) play a key role in regulation of seasonal as well as acute changes in metabolism. Djungarian hamsters (Phodopus sungorus) adapt to winter by multiple changes in behaviour and physiology including spontaneous daily torpor, a state of hypometabolism and hypothermia. We investigated effects of systemic TH administration and ablation on the torpor behaviour in Djungarian hamsters adapted to short photoperiod. Hyperthyroidism was induced by giving T4 or T3 and hypothyroidism by giving methimazole (MMI) and sodium perchlorate via drinking water. T3 treatment increased water, food intake and body mass, whereas MMI had the opposite effect. Continuous recording of body temperature revealed that low T3 serum concentrations increased torpor incidence, lowered Tb and duration, whereas high T3 serum concentrations inhibited torpor expression. Gene expression of deiodinases (dio) and uncoupling proteins (ucp) were analysed by qPCR in hypothalamus, brown adipose tissue (BAT) and skeletal muscle. Expression of dio2, the enzyme generating T3 by deiodination of T4, and ucps, involved in thermoregulation, indicated a tissue specific response to treatment. Torpor per se decreased dio2 expression irrespective of treatment or tissue, suggesting low intracellular T3 concentrations during torpor. Down regulation of ucp1 and ucp3 during torpor might be a factor for the inhibition of BAT thermogenesis. Hypothalamic gene expression of neuropeptide Y, propopiomelanocortin and somatostatin, involved in feeding behaviour and energy balance, were not affected by treatment. Taken together our data indicate a strong effect of thyroid hormones on torpor, suggesting that lowered intracellular T3 concentrations in peripheral tissues promote torpor.

FoxO3a-mediated activation of stress responsive genes during early torpor in a mammalian hibernator.

The torpor-arousal cycle of mammalian hibernation is characterized by drastic changes in physiological state that are supported by reprogramming of metabolic functions. The entrance and arousal phases of the cycle function as transitional stages, where major changes in oxygen metabolism take place. Acute changes in oxygen delivery can lead to either ischemia-related injuries during torpor induction or reperfusion damage during arousal. This study examines the regulation of the forkhead box O3 (FoxO3) transcription factor, which functions to increase cellular cytoprotection in response to oxidative stress stimuli. Immunoblots show that total expression of FoxO3a was elevated during early torpor (ET) and late torpor by 3.6- and 4.5-fold, respectively, compared to euthermic control. However, enhanced phosphorylation of FoxO3a at Thr-32 was only evident during ET by 1.5-fold, accompanied by increased phosphorylation of c-Jun N-terminal kinases by 1.2-fold. During ET, increased nuclear inclusion of FoxO3a was evident along with its transcriptional co-activator ß-catenin by 1.9- and 2.7-fold, respectively. As well, FoxO3a DNA binding was elevated by 1.8-fold during ET, along with increased expression of FoxO3a downstream genes catalase, p27, and cyclin G 2 , by 1.4-, 1.6-, and 1.3-fold, respectively. Overall, the results indicate activation of FoxO3a during ET, suggesting a role of FoxO3a in response to cellular stress during hibernation.

Comparative transcriptomics of the garden dormouse hypothalamus during hibernation.

Torpor or heterothermy is an energy-saving mechanism used by endotherms to overcome harsh environmental conditions. During winter, the garden dormouse (Eliomys quercinus) hibernates with multiday torpor bouts and body temperatures of a few degrees Celsius, interrupted by brief euthermic phases. This study investigates gene expression within the hypothalamus, the key brain area controlling energy balance, adding information on differential gene expression potentially relevant to orchestrate torpor. A de novo assembled transcriptome of the hypothalamus was generated from garden dormice hibernating under constant darkness without food and water at 5 °C. Samples were collected during early torpor, late torpor, and interbout arousal. During early torpor, 765 genes were differentially expressed as compared with interbout arousal. Twenty-seven pathways were over-represented, including pathways related to hemostasis, extracellular matrix organization, and signaling of small molecules. Only 82 genes were found to be differentially expressed between early and late torpor, and no pathways were over-represented. During late torpor, 924 genes were differentially expressed relative to interbout arousal. Despite the high number of differentially expressed genes, only 10 pathways were over-represented. Of these, eight were also observed to be over-represented when comparing early torpor and interbout arousal. Our results are largely consistent with previous findings in other heterotherms. The addition of a transcriptome of a novel species may help to identify species-specific and overarching torpor mechanisms through future species comparisons.

Identification of shared gene expression programs activated in multiple modes of torpor across vertebrate clades.

Torpor encompasses diverse adaptations to extreme environmental stressors such as hibernation, aestivation, brumation, and daily torpor. Here we introduce StrokeofGenus, an analytic pipeline that identifies distinct transcriptomic states and shared gene expression patterns across studies, tissues, and species. We use StrokeofGenus to study multiple and diverse forms of torpor from publicly-available RNA-seq datasets that span eight species and two classes. We identify three transcriptionally distinct states during the cycle of heterothermia: euthermia, torpor, and interbout arousal. We also identify torpor-specific gene expression patterns that are shared both across tissues and between species with over three hundred million years of evolutionary divergence. We further demonstrate the general sharing of gene expression patterns in multiple forms of torpor, implying a common evolutionary origin for this process. Although here we apply StrokeofGenus to analysis of torpor, it can be used to interrogate any other complex physiological processes defined by transient transcriptomic states.

Comparative transcriptomics of the Djungarian hamster hypothalamus during short photoperiod acclimation and spontaneous torpor.

The energy-saving strategy of Djungarian hamsters (Phodopus sungorus, Cricetidae) to overcome harsh environmental conditions comprises of behavioral, morphological, and physiological adjustments, including spontaneous daily torpor, a metabolic downstate. These acclimatizations are triggered by short photoperiod and orchestrated by the hypothalamus. Key mechanisms of long-term photoperiodic acclimatizations have partly been described, but specific mechanisms that acutely control torpor remain incomplete. Here, we performed comparative transcriptome analysis on hypothalamus of normometabolic hamsters in their summer- and winter-like state to enable us to identify changes in gene expression during photoperiodic acclimations. Comparing nontorpid and torpid hamsters may also be able to pin down mechanisms relevant for torpor control. A de novo assembled transcriptome of the hypothalamus was generated from hamsters acclimated to long photoperiod or to short photoperiod. The hamsters were sampled either during long photoperiod normothermia, short photoperiod normothermia, or short photoperiod-induced spontaneous torpor with a body temperature of 24.6 ± 1.0 °C, or. The mRNA-seq analysis revealed that 32 and 759 genes were differentially expressed during photoperiod or torpor, respectively. Biological processes were not enriched during photoperiodic acclimatization but were during torpor, where transcriptional and metabolic processes were reinforced. Most extremely regulated genes (those genes with |log2(FC)| > 2.0 and padj < 0.05 of a pairwise group comparison) underpinned the role of known key players in photoperiodic comparison, but these genes exhibit adaptive and protective adjustments during torpor. Targeted analyses of genes from potentially involved hypothalamic systems identified gene regulation of previously described torpor-relevant systems and a potential involvement of glucose transport.

Integrative transcription start site analysis and physiological phenotyping reveal torpor-specific expression program in mouse skeletal muscle.

Mice enter an active hypometabolic state, called daily torpor when they experience a lowered caloric intake under cold ambient temperature. During torpor, the oxygen consumption rate in some animals drops to less than 30% of the normal rate without harming the body. This safe but severe reduction in metabolism is attractive for various clinical applications; however, the mechanism and molecules involved are unclear. Therefore, here we systematically analyzed the gene expression landscape on the level of the RNA transcription start sites in mouse skeletal muscles under various metabolic states to identify torpor-specific transcribed regulatory patterns. We analyzed the soleus muscles from 38 mice in torpid and non-torpid conditions and identified 287 torpor-specific promoters out of 12,862 detected promoters. Furthermore, we found that the transcription factor ATF3 is highly expressed during torpor deprivation and its binding motif is enriched in torpor-specific promoters. Atf3 was also highly expressed in the heart and brown adipose tissue during torpor and systemically knocking out Atf3 affected the torpor phenotype. Our results demonstrate that mouse torpor combined with powerful genetic tools is useful for studying active hypometabolism.

Profiling torpor-responsive microRNAs in muscles of the hibernating primate Microcebus murinus.

When food scarcity is coupled with decreased temperatures, gray mouse lemurs (Microcebus murinus) depress their metabolic rates and retreat into bouts of either daily torpor or multi-day hibernation, without dramatically dropping body temperatures like other 'traditional hibernators'. Rapid and reversible mechanisms are required to coordinate the simultaneous suppression of energetically expensive processes and activation of pro-survival pathways critical for successful torpor-arousal cycling. MicroRNAs, a class of endogenous non-coding small RNAs, are effective post-transcriptional regulators that modulate all aspects of cellular function. The present study hypothesizes that miRNAs are intimately involved in facilitating the molecular reorganization events necessary for lemur skeletal muscle torpor. Small RNA-Sequencing was used to compare miRNA profiles from skeletal muscles of torpid and control primates. We characterized 234 conserved miRNAs, of which 20 were differentially expressed during torpor, relative to control. Examples included downregulation of key muscle-specific (myomiR) members, miR-1 and miR-133, suggesting a switch to muscle-specific energy-saving strategies. In silico target mapping and logistic regression-based gene set analysis indicated the inhibition of energy costly pathways such as oxidative phosphorylation and muscle proliferation. The suppression of these metabolic pathways was balanced with a lack of miRNA inhibition of various signaling pathways, such as MAPK, mTOR, focal adhesion, and ErbB. This study identifies unique miRNA signatures and 'biomarkers of torpor' that provide us with primate-specific insights on torpor at high body temperatures that can be exploited for human biomedical concerns.

Cold-inducible RNA-binding protein Cirp, but not Rbm3, may regulate transcript processing and protection in tissues of the hibernating ground squirrel.

RNA-binding proteins (RBPs) have important roles in transcription, pre-mRNA processing/transport, mRNA degradation, translation, and non-coding RNA processing, among others. RBPs that are expressed in response to cold stress, such as Cirp and Rbm3, could regulate RNA stability and translation in hibernating mammals that reduce their body temperatures from 37 °C to as low as 0-5 °C during torpor bouts. RBPs including Cirp, Rbm3, and stress-inducible HuR translocate from the nucleus to stabilize mRNAs in the cytoplasm, and thereby could regulate which mRNA transcripts are protected from degradation and are translated, versus stored, for future protein synthesis or degraded by nucleases during cell stress associated with metabolic rate depression. This is the first study to explore the transcriptional/translational regulation, and subcellular localization of cold-inducible RBPs in a model hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus). Cirp protein levels were upregulated in liver, skeletal muscle, and brown adipose tissue throughout the torpor-arousal cycle whereas Rbm3 protein levels stayed constant or decreased, suggesting an important role for Cirp, but likely not Rbm3, in the hibernator stress response. Increased cytoplasmic localization of Cirp in liver and muscle and HuR in liver during torpor, but no changes in the relative levels of Rbm3 in the cytoplasm, emphasizes a role for Cirp and possibly HuR in regulating mRNA processing during torpor. This study informs our understanding of the natural adaptations that extreme animals use in the face of stress, and highlight natural stress response mediators that could be used to bolster cryoprotection of human organs donated for transplant.

Identification of novel and conserved microRNA and their expression in the gray mouse lemur, Microcebus murinus, a primate capable of daily torpor.

MicroRNA (miRNA) are endogenous small noncoding RNA gene products, on average 22 nt long, that play important regulatory roles in mediating gene expression by binding to and targeting mRNAs for degradation or translational repression. In this paper we identify both novel and conserved miRNA sequences present in the genome of the gray mouse lemur, Microcebus marinus. In total, 122 conserved and 44 novel miRNA were identified with high confidence from the lemur genome (Mmur_2.0) and were used for expression analysis. All conserved and novel miRNA were subjected to relative quantification by RT-qPCR in liver samples from control and torpid lemurs. A total of 26 miRNA (16 conserved and 10 novel) showed increased levels during primate torpor, whereas 31 (30 conserved and 1 novel) decreased. Additional in silico mapping of the predicted mRNA targets of torpor-responsive mature miRNA suggested that miRNA that increased during torpor were collectively involved in cell development and survival pathways, while miRNA that decreased were enriched in targeting immune function. Overall, the study suggests new regulatory mechanisms of primate torpor via miRNA action.

Comparative tissue transcriptomics highlights dynamic differences among tissues but conserved metabolic transcript prioritization in preparation for arousal from torpor.

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart, and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.