miRNA Population Research Articles

An ALS-associated mutation dysregulates microglia-derived extracellular microRNAs in a sex-specific manner.

Evidence suggests the presence of microglial activation and microRNA (miRNA) dysregulation in amyotrophic lateral sclerosis (ALS), the most common form of adult motor neuron disease. However, few studies have investigated whether the miRNA dysregulation originates from microglia. Furthermore, TDP-43 (encoded by TARDBP), involved in miRNA biogenesis, aggregates in tissues of ∼98% of ALS cases. Thus, this study aimed to determine whether expression of the ALS-linked TDP-43M337V mutation in a transgenic mouse model dysregulates microglia-derived miRNAs. RNA sequencing identified several dysregulated miRNAs released by transgenic microglia and a differential miRNA release by lipopolysaccharide-stimulated microglia, which was more pronounced in cells from female mice. We validated the downregulation of three candidate miRNAs, namely, miR-16-5p, miR-99a-5p and miR-191-5p, by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and identified their predicted targets, which primarily include genes involved in neuronal development and function. These results suggest that altered TDP-43 function leads to changes in the miRNA population released by microglia, which may in turn be a source of the miRNA dysregulation observed in the disease. This has important implications for the role of neuroinflammation in ALS pathology and could provide potential therapeutic targets.

Identification of circulatory microRNA based biomarkers for early pregnancy diagnosis in buffalo.

Introduction: The most crucial factor in improving animal reproduction efficiency is early pregnancy diagnosis. Early diagnosis not only reduces the time interval between two calvings but also aids farmers in identifying open animals, thereby preventing significant milk production losses. Therefore, the objective of this study was to discover circulatory miRNAs that would be useful for early pregnancy diagnosis in buffalo. Material and methods: Blood samples were taken on 0, 6th, 12th, and 18th day after artificial insemination from pregnant animals (n = 30) and non-pregnant animals (n = 20). During these stages of pregnancy, total RNA was extracted, and a small RNA library was subsequently generated and sequenced on the Illumina platform. Subsequently, Real-time PCR was used to validate the findings. Results and discussion: There were 4,022 miRNAs found during the pregnancy, with 15 of those lacking sequences and 4,007 having sequences already in the database. From the beginning of pregnancy until the 18th day, 25 of these miRNAs showed a substantial shift in expression levels in the maternal blood, with a change more than two logs. Furthermore, based on qPCR results, 19 miRNAs were found to be more abundant in pregnant animals than in non-pregnant animals. We used target prediction analysis to learn how maternally expressed miRNAs relate to fetal-maternal communication. In conclusion, miRNA based biomarkers that could be associated with the diagnosis of pregnancy were identified including miR-181a and miR-486 highly upregulated on the 18th day of pregnancy. This study also provides a comprehensive profile of the entire miRNA population in maternal buffalo blood during the early stages of pregnancy.

Proteomic and genomic profiling of plasma exosomes from patients with ankylosing spondylitis

IntroductionRecent advances in understanding the biology of ankylosing spondylitis (AS) using innovative genomic and proteomic approaches offer the opportunity to address current challenges in AS diagnosis and management. Altered expression...

Intestinal microRNAs and bacterial taxa in juvenile mice are associated, modifiable by allochthonous lactobacilli, and affect postnatal maturation.

The interplay between the intestinal microbiota and host is critical to intestinal ontogeny and homeostasis. MicroRNAs (miRNAs) may be an underlying link. Intestinal miRNAs are microbiota-dependent and, when shed in the lumen, affect resident microorganisms. Yet, longitudinal relationships between intestinal tissue miRNAs, luminal miRNAs, and luminal microorganisms have not been elucidated, especially in early life. Here, we investigated the postnatal cecal miRNA and microbiota populations, their relationship, and their impact on intestinal maturation in specific pathogen-free mice; we also assessed if they can be modified by intervention with allochthonous probiotic lactobacilli. We report that cecal and cecal content miRNA and microbiota signatures are temporally regulated, correlated, and modifiable by probiotics with implications for intestinal maturation. These findings help understand causal relationships within the gut ecosystem and provide a basis for preventing and managing their alterations in diseases throughout life. IMPORTANCE The gut microbiota affects intestinal microRNA (miRNA) signatures and is modified by host-derived luminal miRNA. This suggests the existence of close miRNA-microbiota relationships that are critical to intestinal homeostasis. However, an integrative analysis of these relationships and their evolution during intestinal postnatal maturation is lacking. We provide a system-level longitudinal analysis of miRNA-microbiota networks in the intestine of mice at the weaning transition, including tissue and luminal miRNA and luminal microbiota. To address causality and move toward translational applications, we used allochthonous probiotic lactobacilli to modify these longitudinal relationships and showed that they are critical for intestinal maturation in early life. These findings contribute to understand mechanisms that underlie the maturation of the intestinal ecosystem and suggest that interventions aiming at maintaining, or restoring, homeostasis cannot prescind from considering relationships among its components.

MicroRNA (miRNA) Complexity in Alzheimer's Disease (AD).

AD is a complex, progressive, age-related neurodegenerative disorder representing the most common cause of senile dementia and neurological dysfunction in our elderly domestic population. The widely observed heterogeneity of AD is a reflection of the complexity of the AD process itself and the altered molecular-genetic mechanisms operating in the diseased human brain and CNS. One of the key players in this complex regulation of gene expression in human pathological neurobiology are microRNAs (miRNAs) that, through their actions, shape the transcriptome of brain cells that normally associate with very high rates of genetic activity, gene transcription and messenger RNA (mRNA) generation. The analysis of miRNA populations and the characterization of their abundance, speciation and complexity can further provide valuable clues to our molecular-genetic understanding of the AD process, especially in the sporadic forms of this common brain disorder. Current in-depth analyses of high-quality AD and age- and gender-matched control brain tissues are providing pathophysiological miRNA-based signatures of AD that can serve as a basis for expanding our mechanistic understanding of this disorder and the future design of miRNA- and related RNA-based therapeutics. This focused review will consolidate the findings from multiple laboratories as to which are the most abundant miRNA species, both free and exosome-bound in the human brain and CNS, which miRNA species appear to be the most prominently affected by the AD process and review recent developments and advancements in our understanding of the complexity of miRNA signaling in the hippocampal CA1 region of AD-affected brains.

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance.

FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.

Identification and characterization of miRNAome and target genes in Pseudostellaria heterophylla.

miRNAs play a crucial role in the development and growth of plants by inhibiting the function of targeted genes at the post-transcription level. However, no miRNAs in Pseudostellaria heterophylla have been reported and their function in the morphogenesis of organs is still unclear. In this study, a total of 159 conserved miRNAs (belonging to 64 families) and 303 level miRNAs were identified from P. heterophylla. Some of them showed specifically up or down-regulated expression in different tissues and numbers of unigenes involved in Plant-pathogen interaction and MAPK signaling pathway-plant were targeted. The significant negative correlation of expression profiles between 30 miRNAs and their target genes (37 unigenes) was observed, respectively. Further, a large number of genes involved with signal transduction of auxin, zeatin, abscisic acid and, jasmonic acid were targeted. Predicated targets of two miRNAs were validated by 5′RLM-RACE, respectively. A large number of mRNAs from four pathogens were targeted by miRNAs from P. heterophylla and some of them were targeted by miR414. In summary, we reported a population of miRNAs from four different vegetative tissues of P. heterophylla by high throughput sequencing, which was analyzed by combining with the constructed transcriptome. These results may help to explain the function of miRNAs in the morphogenesis of organs and defense of pathogens, and may provide theoretical basis for breeding and genetic improvement of P. heterophylla.

Synaptosome microRNAs regulate synapse functions in Alzheimer’s disease

MicroRNAs (miRNAs) are found in nerve terminals, synaptic vesicles, and synaptosomes, but it is unclear whether synaptic and cytosolic miRNA populations differ in Alzheimer’s disease (AD) or if synaptosomal miRNAs affect AD synapse activity. To address these questions, we generated synaptosomes and cytosolic fractions from postmortem brains of AD and unaffected control (UC) samples and analyzed them using a global Affymetrix miRNAs microarray platform. A group of miRNAs significantly differed (P < 0.0001) with high fold changes variance (+/− >200-fold) in their expressions in different comparisons: (1) UC synaptosome vs UC cytosol, (2) AD synaptosomes vs AD cytosol, (3) AD cytosol vs UC cytosol, and (4) AD synaptosomes vs UC synaptosomes. MiRNAs data analysis revealed that some potential miRNAs were consistently different across sample groups. These differentially expressed miRNAs were further validated using AD postmortem brains, brains of APP transgenic (Tg2576), Tau transgenic (P301L), and wild-type mice. The miR-501-3p, miR-502-3p, and miR-877-5p were identified as potential synaptosomal miRNAs upregulated with disease progression based on AD Braak stages. Gene Ontology Enrichment and Ingenuity Pathway Analysis of synaptosomal miRNAs showed the involvement of miRNAs in nervous system development, cell junction organization, synapse assembly formation, and function of GABAergic synapse. This is the first description of synaptic versus cytosolic miRNAs in AD and their significance in synapse function.

A comparative study of microRNAs in different stages of Eimeria tenella

Apicomplexan parasites have divergent biogenesis machinery for small RNA generation. Analysis has shown that parasites in Plasmodium and Cryptosporidium as well as many species in Leishmania or Trypanosoma do not have a complete machinery in small RNA biogenesis. Recently, the miRNA-generating system of Toxoplasma has been identified as plant/fungal-like and its miRNAome has been elucidated. However, the microRNA (miRNA) expression profiles and their potential regulatory functions in different stages of Eimeria tenella remain largely unknown. In this study, we characterized the RNA silencing machinery of E. tenella and investigated the miRNA population distribution at different life stages by high-throughput sequencing. We characterized the expression of miRNAs in the unsporulated oocyst, sporulated oocyst and schizogony stages, obtaining a total of 392 miRNAs. We identified 58 differentially expressed miRNAs between USO (unsporulated oocysts) and SO (sporulated oocysts) that were significantly enriched for their potential target genes in the regulation of gene expression and chromatin binding, suggesting an epigenetic modulation of sporulating by these miRNAs. In comparing miRNA expression at endogenous and exogenous developmental stages, twenty-four miRNAs were identified differently expressed. Those were mainly associated with the regulation of genes with protein kinase activity, suggesting control of protein phosphorylation. This is the first study about the evolution of miRNA biogenesis system and miRNA control of gene expression in Eimeria species. Our data may lead to functional insights into of the regulation of gene expression during parasite life cycle in apicomplexan parasites.

Parasitic plant small RNA analyses unveil parasite-specific signatures of microRNA retention, loss, and gain.

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that C. campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic O. aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

Characteristics of miRNAs Present in Bovine Sperm and Associations With Differences in Fertility.

Small non-coding RNAs have been linked to different phenotypes in bovine sperm, however attempts to identify sperm-borne molecular biomarkers of male fertility have thus far failed to identify a robust profile of expressed miRNAs related to fertility. We hypothesized that some differences in bull fertility may be reflected in the levels of different miRNAs in sperm. To explore such differences in fertility that are not due to differences in visible metrics of sperm quality, we employed Next Generation Sequencing to compare the miRNA populations in Bos taurus sperm from bulls with comparable motility and morphology but varying Sire Conception Rates. We identified the most abundant miRNAs in both populations (miRs -34b-3p; -100-5p; -191-5p; -30d-4p; -21-5p) and evaluated differences in the overall levels and specific patterns of isomiR expression. We also explored correlations between specific pairs of miRNAs in each population and identified 10 distinct pairs of miRNAs that were positively correlated in bulls with higher fertility and negatively correlated in comparatively less fertile individuals. Furthermore, 8 additional miRNA pairs demonstrated the opposite trend; negatively correlated in high fertility animals and positively correlated in less fertile bulls. Finally, we performed pathway analysis to identify potential roles of miRNAs present in bull sperm in the regulation of specific genes that impact spermatogenesis and embryo development. Together, these results present a comprehensive picture of the bovine sperm miRNAome that suggests multiple potential roles in fertility.

SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2-infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). Its abundance ranges from low to moderate as compared to host miRNAs and it associates with Argonaute proteins-core components of the RNA interference pathway. We identify putative targets for CoV2-miR-O7a, including Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which participates in interferon signaling. We demonstrate that CoV2-miR-O7a production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.

Integration of mRNA and miRNA Analysis Reveals the Molecular Mechanism of Cotton Response to Salt Stress.

To identify the regulatory network of known and novel microRNAs (miRNAs) and their targets responding to salt stress, a combined analysis of mRNA libraries, small RNA libraries, and degradome libraries were performed. In this study, we used unique molecular identifiers (UMIs), which are more sensitive, accurate, and reproducible than traditional methods of sequencing, to quantify the number of molecules and correct for amplification bias. We identified a total of 312 cotton miRNAs using seedlings at 0, 1, 3, and 6 h after NaCl treatment, including 80 known ghr-miRNAs and 232 novel miRNAs and found 155 miRNAs that displayed significant differential expression under salt stress. Among them, fifty-nine differentially expressed miRNAs were simultaneously induced in two or three tissues, while 66, 11, and 19 were specifically expressed in the roots, leaves, and stems, respectively. It is indicated there were different populations of miRNAs against salt stress in roots, leaves and stems. 399 candidate targets of salt-induced miRNAs showed significant differential expression before and after salt treatment, and 72 targets of 25 miRNAs were verified by degradome sequencing data. Furthermore, the regulatory relationship of miRNA-target gene was validated experimentally via 5′RLM-RACE, proving our data reliability. Gene ontology and KEGG pathway analysis found that salt-responsive miRNA targets among the differentially expressed genes were significantly enriched, and mainly involved in response to the stimulus process and the plant hormone signal transduction pathway. Furthermore, the expression levels of newly identified miRNA mir1 and known miRNAs miR390 and miR393 gradually decreased when subjected to continuous salt stress, while overexpression of these miRNAs both increased sensitivity to salt stress. Those newly identified miRNAs and mRNA pairs were conducive to genetic engineering and better understanding the mechanisms responding to salt stress in cotton.

Expression analysis of miRNAs and their putative target genes confirm a preponderant role of transcription factors in the early response of oil palm plants to salinity stress

BackgroundSeveral mechanisms regulating gene expression contribute to restore and reestablish cellular homeostasis so that plants can adapt and survive in adverse situations. MicroRNAs (miRNAs) play roles important in the transcriptional and post-transcriptional regulation of gene expression, emerging as a regulatory molecule key in the responses to plant stress, such as cold, heat, drought, and salt. This work is a comprehensive and large-scale miRNA analysis performed to characterize the miRNA population present in oil palm (Elaeis guineensis Jacq.) exposed to a high level of salt stress, to identify miRNA-putative target genes in the oil palm genome, and to perform an in silico comparison of the expression profile of the miRNAs and their putative target genes.ResultsA group of 79 miRNAs was found in oil palm, been 52 known miRNAs and 27 new ones. The known miRNAs found belonged to 28 families. Those miRNAs led to 229 distinct miRNA-putative target genes identified in the genome of oil palm. miRNAs and putative target genes differentially expressed under salinity stress were then selected for functional annotation analysis. The regulation of transcription, DNA-templated, and the oxidation-reduction process were the biological processes with the highest number of hits to the putative target genes, while protein binding and DNA binding were the molecular functions with the highest number of hits. Finally, the nucleus was the cellular component with the highest number of hits. The functional annotation of the putative target genes differentially expressed under salinity stress showed several ones coding for transcription factors which have already proven able to result in tolerance to salinity stress by overexpression or knockout in other plant species.ConclusionsOur findings provide new insights into the early response of young oil palm plants to salinity stress and confirm an expected preponderant role of transcription factors - such as NF-YA3, HOX32, and GRF1 - in this response. Besides, it points out potential salt-responsive miRNAs and miRNA-putative target genes that one can utilize to develop oil palm plants tolerant to salinity stress.

Distinct Exosomal miRNA Profiles from BALF and Lung Tissue of COPD and IPF Patients.

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic, progressive lung ailments that are characterized by distinct pathologies. Early detection biomarkers and disease mechanisms for these debilitating diseases are lacking. Extracellular vesicles (EVs), including exosomes, are small, lipid-bound vesicles attributed to carry proteins, lipids, and RNA molecules to facilitate cell-to-cell communication under normal and diseased conditions. Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of bronchoalveolar lavage fluid (BALF) or the lung-tissue-derived exosomes in COPD and IPF. Here, we determined and compared the miRNA profiles of BALF- and lung-tissue-derived exosomes of healthy non-smokers, smokers, and patients with COPD or IPF in independent cohorts. Results: Exosome characterization using NanoSight particle tracking and TEM demonstrated that the BALF-derived exosomes were ~89.85 nm in size with a yield of ~2.95 × 1010 particles/mL in concentration. Lung-derived exosomes were larger in size (~146.04 nm) with a higher yield of ~2.38 × 1011 particles/mL. NGS results identified three differentially expressed miRNAs in the BALF, while there was one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, miR-122-5p was three- or five-fold downregulated among the lung-tissue-derived exosomes of COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were a large number (55) of differentially expressed miRNAs in the lung-tissue-derived exosomes of IPF patients compared to non-smoking controls. Conclusions: Overall, we identified lung-specific miRNAs associated with chronic lung diseases that can serve as potential biomarkers or therapeutic targets.

Learning noncoding RNA biology from viruses.

Insights into interactions between viral factors and the cellular machinery usually lead to discoveries concerning host cell biology. Thus, the gene expression field has historically relied on viral model systems to discover mechanisms underlying different cellular processes. In recent years, the functional characterization of the small nuclear noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, called HSURs, resulted in the discovery of two mechanisms for the regulation of gene expression. HSUR1 and HSUR2 associate with host microRNAs, which are small noncoding RNAs that broadly regulate gene expression by binding to messenger RNAs. HSUR1 provided the first example of a process known as target-directed miRNA degradation that operates in cells to regulate miRNA populations. HSUR2 functions as a miRNA adaptor, uncovering an entirely new, indirect mechanism by which miRNAs can inhibit mRNA function. Here, I review the path that led to these discoveries and their implications and postulate new exciting questions about the functions of these fascinating viral noncoding RNAs.

Molecular underpinnings of the early brain developmental response to differential feeding in the honey bee Apis mellifera

Brain differential morphogenesis in females is one of the major phenotypic manifestations of caste development in honey bees. Brain diphenism appears at the fourth larval phase as a result of the differential feeding regime developing females are submitted during early phases of larval development. Here, we used a forward genetics approach to test the early brain molecular response to differential feeding leading to the brain diphenism observed at later developmental phases. Using RNA sequencing analysis, we identified 53 differentially expressed genes (DEGs) between the brains of queens and workers at the third larval phase. Since miRNAs have been suggested to play a role in caste differentiation after horizontal and vertical transmission, we tested their potential participation in regulating the DEGs. The miRNA-mRNA interaction network, including the DEGs and the royal- and worker-jelly enriched miRNA populations, revealed a subset of miRNAs potentially involved in regulating the expression of DEGs. The interaction of miR-34, miR-210, and miR-317 with Takeout, Neurotrophin-1, Forked, and Masquerade genes was experimentally confirmed using a luciferase reporter system. Taken together, our results reconstruct the regulatory network that governs the development of the early brain diphenism in honey bees.

Genome-Wide Analysis of Coding and Non-coding RNA Reveals a Conserved miR164-NAC-mRNA Regulatory Pathway for Disease Defense in Populus.

MicroRNAs (miRNAs) contribute to plant defense responses by increasing the overall genetic diversity; however, their origins and functional importance in plant defense remain unclear. Here, we employed Illumina sequencing technology to assess how miRNA and messenger RNA (mRNA) populations vary in the Chinese white poplar (Populus tomentosa) during a leaf black spot fungus (Marssonina brunnea) infection. We sampled RNAs from infective leaves at conidia germinated stage [12 h post-inoculation (hpi)], infective vesicles stage (24 hpi), and intercellular infective hyphae stage (48 hpi), three essential stages associated with plant colonization and biotrophic growth in M. brunnea fungi. In total, 8,938 conserved miRNA-target gene pairs and 3,901 Populus-specific miRNA-target gene pairs were detected. The result showed that Populus-specific miRNAs (66%) were more involved in the regulation of the disease resistance genes. By contrast, conserved miRNAs (>80%) target more whole-genome duplication (WGD)-derived transcription factors (TFs). Among the 1,023 WGD-derived TF pairs, 44.9% TF pairs had only one paralog being targeted by a miRNA that could be due to either gain or loss of a miRNA binding site after the WGD. A conserved hierarchical regulatory network combining promoter analyses and hierarchical clustering approach uncovered a miR164–NAM, ATAF, and CUC (NAC) transcription factor–mRNA regulatory module that has potential in Marssonina defense responses. Furthermore, analyses of the locations of miRNA precursor sequences reveal that pseudogenes and transposon contributed a certain proportion (∼30%) of the miRNA origin. Together, these observations provide evolutionary insights into the origin and potential roles of miRNAs in plant defense and functional innovation.

Does miRNA Expression in the Spent Media Change During Early Embryo Development?

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.

MicroRNA expression variation as a potential molecular mechanism contributing to adaptation to hydrogen sulphide.

microRNAs (miRNAs) are post-transcriptional regulators of gene expression and can play an important role in modulating organismal development and physiology in response to environmental stress. However, the role of miRNAs in mediating adaptation to diverse environments in natural study systems remains largely unexplored. Here, we characterized miRNAs and their expression in Poecilia mexicana, a species of small fish that inhabits both normal streams and extreme environments in the form of springs rich in toxic hydrogen sulphide (H2 S). We found that P.mexicana has a similar number of miRNA genes as other teleosts. In addition, we identified a large population of mature miRNAs that were differentially expressed between locally adapted populations in contrasting habitats, indicating that miRNAs may contribute to P.mexicana adaptation to sulphidic environments. In silico identification of differentially expressed miRNA-mRNA pairs revealed, in the sulphidic environment, the downregulation of miRNAs predicted to target mRNAs involved in sulphide detoxification and cellular homeostasis, which are pathways essential for life in H2 S-rich springs. In addition, we found that predicted targets of upregulated miRNAs act in the mitochondria (16.6% of predicted annotated targets), which is the main site of H2 S toxicity and detoxification, possibly modulating mitochondrial function. Together, the differential regulation of miRNAs between these natural populations suggests that miRNAs may be involved in H2 S adaptation by promoting functions needed for survival and reducing functions affected by H2 S. This study lays the groundwork for further research to directly demonstrate the role of miRNAs in adaptation to H2 S. Overall, this study provides a critical stepping-stone towards a comprehensive understanding of the regulatory mechanisms underlying the adaptive variation in gene expression in a natural system.