microRNA profiling in the Weddell Seal suggests novel regulatory mechanisms contributing to diving adaptation
Background. The Weddell Seal (Leptonychotes weddelli) represents a remarkable example of adaptation to diving among marine mammals. This species is capable of diving >900 m deep and remaining underwater for more than 60 minutes. A number of key physiological specializations have been identified, including the low levels of aerobic, lipid-based metabolism under hypoxia, significant increase in oxygen storage in blood and muscle; high blood volume and extreme cardiovascular control. These
... trol. These adaptations have been linked to increased abundance of key proteins, suggesting an important, yet still understudied role for gene reprogramming. In this study, we investigate the possibility that post-transcriptional gene regulation by microRNAs (miRNAs) has contributed to the adaptive evolution of diving capacities in the Weddell Seal. Results. Using small RNA data across 4 tissues (cortex, heart, muscle and plasma), in 3 biological replicates, we generate the first miRNA annotation in this species, consisting of 559 high confidence, manually curated miRNA loci. Evolutionary analyses of miRNA gain and loss highlight a high number of Weddell seal specific miRNAs. 416 miRNAs were differentially expressed (DE) among tissues, whereas 83 miRNAs were differentially expressed (DE) across all tissues between pups and adults and 188 miRNAs demonstrated developmental changes in specific tissues only. mRNA targets of these altered miRNAs identify possible protective mechanisms in individual tissues, particularly relevant to hypoxia tolerance, antiapoptotic pathways, and nitric oxide signal transduction. Novel, lineage-specific miRNAs associated with developmental changes target genes with roles in angiogenesis and vasoregulatory signaling. Conclusions. Altogether, we provide an overview of miRNA composition and evolution in the Weddell seal, and the first insights into their possible role in the specialization to diving. Background The Antarctic Weddell Seal (Leptonychotes weddelli) is a deep diving marine mammal, capable of pursuing prey to depths >900 m and remaining underwater for more than 60 minutes [1, 2] . Due to their exceptional diving ability and accessibility on the fast ice during their breeding season, the 3 Weddell seal is one of the best-studied divers in the world. The pinniped lineage recolonized the marine environment ~25 mya  and over this evolutionary time have become specialized to their aquatic habitat. These specializations encompass morphology and physiology; in particular, the extreme cardiovascular physiology of diving mammals is central to their capacity for long-duration diving. The well-developed dive response of marine mammals, including Weddell seals, is characterized by cardiovascular adjustments to lower heart rate and reduce peripheral blood flow during submergence. These adjustments depress tissue oxygen use by restricting its availability to peripheral vascular beds and conserving it for critical central tissues such as the brain and heart. Previous work has also highlighted several complementary traits that support breath-hold hunting in seals, for example: the preference for aerobic, lipid-based metabolism under hypoxia [4-6]; and extremely high oxygen stores in blood and muscle via enhanced haemoglobin and myoglobin [7-9]. Pinnipeds provide a fascinating model system in which to study the development of diving ability and hypoxia tolerance in mammals. Only adult seals are elite divers -unlike cetacean calves, pinniped pups are born on land. Development of the adult diving phenotype has been linked to changes in key proteins (e.g. respiratory pigments), tissue iron content and metabolic enzyme levels . However, the details of the extent of tissue-specific maturation to refine local blood flow , metabolic control, and to combat negative effects of hypoxia exposure are still to be elucidated. Interestingly, pup physiology develops during weaning and throughout a post-weaning fast, including cardiac ontogeny to develop the fine-scale control of bradycardia observed in adults. This maturation can begin before pups first enter the water, which suggests an important role for gene reprogramming. The contribution of post-transcriptional gene regulation in the development of hypoxia tolerance and dive capacity has not been investigated. MicroRNAs (miRNAs) are considered one of the key gene regulators in animals, conferring temporospatial precision in the regulation of gene expression. These short (~22 nt) non-coding RNAs are involved in fundamental processes such as embryonic development and tissue differentiation  and likely play important roles in seasonal and developmental transitions involving gene reprogramming. MiRNAs reduce translation by binding to the 3' region of complementary RNA, Recent studies have highlighted the high rate of novel miRNA gains in mammals . We identified 874 loci clusters in a dataset that included Weddell seal, cow, dog, horse, pig, rabbit, mouse, and human. These were considered miRNA orthogroups and were evaluated to infer the evolutionary patterns of gain and loss across the phylogenetic tree (Fig. 4) . High net gain rates in both the dog and the Weddell Seal lineages suggest dynamic miRNA evolution. For the seal, we also observe a high number of lineage specific losses. 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