MicroRNA profiling in the Weddell seal suggests novel regulatory mechanisms contributing to diving adaptation

- 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.
- 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..
- Evolutionary analyses of miRNA gain and loss highlight a high number of Weddell seal specific miRNAs..
- 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..
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- Due to their exceptional diving ability and accessibility on the fast ice during their breeding season, the Weddell seal is one of the best-studied divers in the world.
- Development of the adult diving phenotype has been linked to changes in key proteins (e.g.
- However, the details of the extent of tissue-specific maturation to refine local blood flow [10], metabolic control, and to combat negative effects of hypoxia ex- posure are still to be elucidated.
- 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 temporo-spatial precision in the regulation of gene expression.
- For example, differential miRNA profiles in the highland yak compared to the lowland cow are enriched for hyp- oxia signaling pathways in the respiratory and cardiovas- cular systems – a key component of altitude adaptation [20].
- In this study, we investigate a potential role for miRNAs in Weddell seal maturation, by post-transcriptional regu- lation of genes involved in development of the dive re- sponse and hypoxia tolerance.
- These results illuminated a large portion of the read data that was unmapped to the Wed- dell seal genome (Additional file 1: Fig.
- This sequence did not map to the Weddell seal genome, but was identified as miR-143-3p.
- Thus, assembly incompleteness appears to reduce the percentage of small RNA reads that could be mapped to the Weddell seal genome, with the greatest impact on plasma samples..
- We examined variation in the relative abundance of miRNA-5p and miRNA-3p across all samples and did not identify any clear examples of arm switching (i.e..
- changes of the most abundant miRNA strand)..
- 50% of tissue-specific DE occurred in the brain, with 31% of significant results in plasma.
- miR-499-5p was the most DE locus in the dataset, a Random Forests classifier, and has the highest expres- sion in heart (Additional file 3: Table S5).
- miR-490-3p was also highly expressed in the heart, was a Random Forests classifier, and was among the five most significant DE changes in the data- set (Additional file 3: Table S6).
- miR-490-3p targets ion channels and transporters, including members of the KCN and SLC gene families (Kcng3, Kcnj15, Kcne2, Kcne4, Kcnmb1, Kcnip3, Kcnk12, Slc1A3, Slc9a1, Slc9a2), associ- ated with pathway enrichments in potassium ion trans- membrane transport (GO:0071804, GO:0071805).
- In general, targets of DE miRNAs in the seal brain were enriched for neuronal and developmental processes (Additional file 3: Table S6).
- Of the Random Forest classifiers DE in plasma (Table 2), miR-95-3p had the largest sample-specific downregu- lation (20-53X lower than heart, muscle, and brain.
- Gradient of blue corresponds to distance, while samples are color coded based on the tissue origin and developmental stage of the individual.
- Colour labels are based on both the tissue origin and developmental stage of the individual.
- Only 13% of significant adult-pup differences occurred in the heart and there were no develop- mental differences in plasma miRNAs (Table 1)..
- Predicted targets of miR-29a-3p that would be reduced from a terrestrial pup to a diving adult include compo- nents of the mitochondrial electron transport system.
- Conversely, higher miR- 542-5p in pups is predicted to enhance the abundance of mRNAs that are likely relevant in the adult pheno- type.
- Novel Weddell seal miRNAs are more likely to be tissue-specific, as we observed a significantly higher pro- portion of tissue-specific miRNA families in the novel set compared to the miRBase set (z test, brain: p = 0.0001;.
- Supervised classification in Random Forests analyses of the four tissue types had zero error, clustering of samples by developmental stage had out of bag error.
- 3 Random Forests plots classifying Weddell seal sample types based on a minimal set of microRNA abundance inputs.
- For example, VEGF signaling was targeted by both novel and annotated miRNAs in the seal heart, highlighting the importance of vascular development in cardiac tissue.
- 0.05, see Methods) for the targets of miRNAs that were highest in the brain and 31 pathways were enriched for targets of muscle-elevated miRNAs, which are primarily signaling pathways.
- Several of these pathways are associated with lipid metabolism (Peroxisome in brain, ABC transporters in heart) and in- flammatory signaling (Cytokine-cytokine receptor and Jak- stat signaling in plasma, CAMs in skeletal muscle), both important elements of the Weddell seal phenotype (Fig.
- Specific novel miRNAs also indicate potential to control key biochemical and physiological features of the Weddell seal.
- Novel-15-3p, highly expressed in the Weddell seal heart, is associated with cardiomyopathy through its target dystrophin.
- Beyond this, we examined the 220 cases of DE between adult and pup in either the brain, heart, or muscle specifically, which may regulate the development of the diving phenotype (Additional file 3: Table S7).
- The main de- velopmental signal linked to hypoxia responses was the expression of miR-424-3p, which is significantly reduced in the heart overall, and additionally downregulated in Table 3 The number of unique mRNA targets of differentially expressed miRNAs in four target tissues, with developmental stage, and in tissue-specific developmental comparisons.
- orthogroups are listed for each branch of the tree (black).
- Differentially upregulated miRNAs target the beta-1 subunit of guanylyl cyclase (Gucy1b1), which would decrease local expression of the heterodimeric enzyme in the brain, heart and muscle overall, whereas miRNAs targeting Gucy1b1 were down- regulated in plasma (Fig.
- Colouring denotes the percent of miRNA families expressed in each tissue that are tissue-specific, expressed in the tissue of interest and one.
- Four of the novel miR- NAs upregulated in the adult brain and 2 that are upregulated in the adult heart are predicted to target Vash1, an angiogenesis inhibitor selective to endothe- lial cells.
- In this study, we provide the first annotation of the Weddell seal miRNome, by manually curating the com- putational predictions of MiRCat2 and MiRDeep2.
- Evolu- tionary and comparative analyses highlight high lineage- specific miRNA gains in the Weddell Seal, compared to other mammals.
- regulated in each tissue compared to all others and categorized as pathways related to previously annotated miRNAs versus novel, unannotated miRNAs identified in the Weddell seal (novel.
- Protective mechanisms relevant to diving in each tissue Differential expression of cardiac miRNAs are the most limited in the dataset but represent some of the largest magnitude changes, which highlight miRNAs related to is- chemic stress.
- In particular, the pro-apoptotic Jade1 target is predicted to be reduced in the seal heart due to high miR- 499-5p, suggesting a protective mechanism against cardiac ischemia [40].
- Activation of Asic3 con- tributes to neurally controlled local vasoconstriction, which would facilitate high peripheral vascular resistance and maintenance of the dive response despite hypoxia..
- Brain and plasma have the most DE among miRNAs, likely to due to the high heterogeneity of tissue types found in the brain, and the contribution of many tissues releasing miRNAs into the venous circulation.
- While the brain is expected to be an enriched target for many hor- mone signaling pathways, it is potentially relevant that the largest DE miRNA in the brain (miR-488-3p) also targets steroid hormone receptors..
- Rows correspond to tissues, columns to the direction of the expression change.
- Groups of miRNAs targeting the same gene in the same DE group are represented by stacked columns, with a height proportional to the number of different miRNAs.
- Adults maintain aerobic metabolism during routine diving by large oxygen storage abilities of the blood and muscle [50–52].
- Downregulation of tran- scripts encoding proteins of the electron transport chain in skeletal muscle with age, predicted from this study, are however consistent with significantly reduced mito- chondrial volume densities in Weddell seal adults com- pared to pups from the same study [59]..
- Indeed, downregulation of the nitric oxide-guanylyl cyclase-cGMP signal transduction pathway is limited in Weddell seals [10], which may facilitate central nervous system control of the dive response, but also reduce pro- duction of harmful peroxynitrites in the presence of re- active oxygen species.
- The number of DE miRNAs targeting this pathway, in particular novel miRNAs, strongly suggest that lineage-specific miRNA regulation contributes to control of local vasoregulators in the Weddell seal, and perhaps the evolution of diving cap- abilities.
- The most significant mRNA target of the path- way in this dataset is NO synthase 1 (Nos1).
- is described as the neuronal form, Nos1 has wide expres- sion across tissues and in additional to functions in the brain, plays a role in systemic control of vascular smooth muscle tone [64] and myocardial function.
- For example, miR-424-3p, which regulates HIF1α through interactions with proteins in the ubiquitin-ligase system, as well as promotes angiogenesis in vitro in mice, is significantly reduced in heart compared to other tissues and further downregulated in the adult heart compared to the pup.
- Similar miRNA targeting of hypoxia sig- naling pathways was highlighted as an adaptation to high altitude in the yak, however a distinct set of miR- NAs of interest were identified [20]..
- Novel, Weddell seal-specific miRNAs are evolution- arily young, and may provide key insights into the evolution of the diving phenotype.
- however, they ap- pear to be under the control of different sets of miRNAs in the 2 species.
- 50% of novel miRNAs which are increased in the adult brain and heart target Vash1, an angiogenesis in- hibitor selective to endothelial cells.
- particularly as they share the potential to regulate the ex- pression of the receptor for endothelin 1..
- Key differences among the tissues are informative towards the biology, but are difficult to interpret in the context of species-specific adaptations without comparison to background(s) of tissue-specific miRNAomes in other species.
- The detec- tion of miRNA targets relied on the 3′ UTR multiple alignments available in the TargetScan7 database [68]..
- Functional confirmation of these interac- tions and their biological roles in the Weddell seal would be important future work towards understanding the molecular basis of the diving phenotype..
- It is also important to acknowledge details of the sam- ple collection which may impact interpretation of the re- sults.
- In this study, we take the first steps towards answering the question of whether post-transcriptional regulation by miRNAs has played a role in the evolution of diving capacities in the Weddell Seal.
- Targets of differentially expressed miRNAs are enriched for GO categories relevant to Weddell seal physiology, suggesting that miRNAs have indeed contributed to the evolution of extreme diving cap- abilities, and providing a new set of mechanistic hypotheses for future follow-ups.
- We also present a thorough miRNA annotation of the Weddell seal genome, representing a valuable resource for future biomedical and evolutionary studies.
- Recent studies in vertebrates have highlighted expression divergence between vertebrate lineages, and the crucial role of gene regulation in the evolution of adaptive phenotypes [70–76].
- It is yet another step towards a wider description of miRNA evolutionary dynamics in mammals, which can fur- ther clarify the contribution of gene regulatory mechanisms in the evolution of adaptive traits..
- We annotated miRNA loci within the Weddell seal gen- ome based on the combined set of small RNA libraries across all tissues and developmental stages, using both miRCat2 [27] and miRDeep2 [28] (Additional file 1: Fig..
- This led to the generation of the final annotation, consisting of 559 high confidence miRNA loci..
- Analysis was implemented using the varSelRF package [83] to eliminate the least important input vari- ables based on 100,000 trees in the first forest and 50, 000 trees in all subsequent iterations.
- We then identified the closest protein coding gene upstream and downstream of the selected hits, as well as the gene containing the hit for all intragenic hits.
- Read statistics considering different stages of the bioinformatic analysis: number of raw reads.
- Selected GO accessions enriched in the targets of miR-339-3p.
- For each locus, the full hairpin sequence is shown, followed by the set of reads (one per line) perfectly matching the locus (with the corresponding abundance) and the predictive secondary structure of the miRNA hairpin..
- Significant pathway enrichments in brain, heart, and muscle for mRNA targets of all microRNAs differentially expressed in Weddell seal maturation.
- in four Weddell seal sample types.
- Number of significant pathway enrichments annotated to mRNA targets of novel Weddell seal microRNAs that were elevated.
- The funding bodies had no role in the design of the study, in the collection, the analysis or the interpretation of data and in writing the manuscript.
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