Genome-Wide and Species-Wide In Silico Screening for Intragenic MicroRNAs in Human,Mouse and Chicken

MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) involved in regulation of gene expression. Intragenic miRNAs, especially those exhibiting a high degree of evolutionary conservation, have been shown to be coordinately regulated and/or expressed with their host genes, either with synergistic or antagonistic correlation patterns. However, the degree of cross-species conservation of miRNA/host gene co-location is not known and co-expression information is incomplete and fragmented among several studies. Using the genomic resources (miRBase and Ensembl) we performed a genome-wide in silico screening (GWISS) for miRNA/host gene pairs in three well-annotated vertebrate species: human, mouse, and chicken. Approximately half of currently annotated miRNA genes resided within host genes: 53.0% (849/1,600) in human, 48.8% (418/855) in mouse, and 42.0% (210/499) in chicken, which we present in a central publicly available Catalog of intragenic miRNAs (http://www.integratomics-time.com/miR-host/catalog). The miRNA genes resided within either protein-coding or ncRNA genes, which include long intergenic ncRNAs (lincRNAs) and small nucleolar RNAs (snoRNAs). Twenty-seven miRNA genes were found to be located within the same host genes in all three species and the data integration from literature and databases showed that most (26/27) have been found to be co-expressed. Particularly interesting are miRNA genes located within genes encoding for miRNA silencing machinery (DGCR8, DICER1, and SND1 in human and Cnot3, Gdcr8, Eif4e, Tnrc6b, and Xpo5 in mouse). We furthermore discuss a potential for phenotype misattribution of miRNA host gene polymorphism or gene modification studies due to possible collateral effects on miRNAs hosted within them. In conclusion, the catalog of intragenic miRNAs and identified 27 miRNA/host gene pairs with cross-species conserved co-location, co-expression, and potential co-regulation, provide excellent candidates for further functional annotation of intragenic miRNAs in health and disease.     

In vitro quantification of specific microRNA using molecular beacons

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identi?cation of 108 miRNAs (out of 1,417; 7.6%) that showed signi?cant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem–loop real time quantitative PCR (RT‐qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3′‐untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.     

Integration of miRNA weighted gene co-expression network and miRNA-mRNA co-expression analyses reveals potential regulatory functions of miRNAs in calf rumen development

This study aimed to explore the roles of microRNAs (miRNAs) in calf rumen development during early life. Rumen tissues were collected from 16 calves (8 at pre-weaning and 8 at post-weaning) for miRNA-sequencing, differential expression (DE), miRNA weighted gene co-expression network (WGCNA) and miRNA-mRNA co-expression analyses. 295 miRNAs were identified. Bta-miR-143, miR-26a, miR-145 and miR-27b were the most abundantly expressed. 122 miRNAs were significantly DE between the pre- and post-weaning periods and the most up- and down-regulated miRNAs were bta-miR-29b and bta-miR-493, respectively. Enrichment analyses of the target genes of DE miRNAs revealed important roles for miRNA in rumen developmental processes, immune system development, protein digestion and processes related to the extracellular matrix. WGCNA indicated that bta-miR-145 and bta-miR-199a-3p are important hub miRNAs in the regulation of these processes. Therefore, bta-miR-143, miR-29b, miR-145, miR-493, miR-26a and miR-199 family members might be key regulators of calf rumen development during early life.     

家蚕5龄幼虫精巢和卵巢microRNA芯片及转录组比较分析

【目的】本研究旨在通过对家蚕Bombyx mori 5龄幼虫精巢和卵巢组织微小RNA (microRNA, miRNA)基因芯片及转录组进行分析,找到参与家蚕性腺发育相关的miRNA分子及可能的靶基因。【方法】采用新一代高通量测序平台对家蚕5龄幼虫精巢和卵巢(分别定义为Test和Control)进行miRNA基因芯片检测及转录组测序分析,根据P＜0.05且log₂(fold change, FC)≥2的标准,通过比较筛选出Test vs Control的差异表达miRNA;根据q≤0.05且|log₂(fold change)|≥1的标准,通过比较筛选出Test vs Control的差异表达基因 (differentially expressed genes, DEGs);随机选取8个上调和12个下调差异表达miRNA,对其表达及其预测的5个靶基因进行qRT-PCR验证;对DEGs以及差异表达miRNA的靶基因进行KEGG通路富集分析。【结果】从精巢和卵巢样本中(Test vs Control)分别鉴定出68个差异表达miRNA和3 991个DEGs,其中上调和下调miRNA分别为36和32个,上调和下调DEGs分别为2 033和1 958个。差异表达miRNA的qRT PCR验证结果均与芯片数据一致。KEGG通路富集分析结果显示DEGs在新陈代谢及核糖体的信号通路显著富集。对差异表达miRNA在DEGs中的可能靶基因进行预测,结果找到了4组表达趋势相反的miRNA与靶基因：分别是bmo-miR-2774a与LOC101745556;bmo-miR-92b与LOC101735954以及bmo-miR-3266与LOC733130和LOC778467;1组表达趋势一致的miRNA与靶基因：bmo-miR-3321与LOC101744895。5个靶基因的qRT-PCR验证结果与转录组测序结果一致。【结论】本研究获得了家蚕5龄幼虫精巢和卵巢转录组及miRNA芯片数据,筛选并验证了4组差异表达和1组一致表达miRNA及潜在靶基因,为探究家蚕精巢和卵巢发育差异奠定了基础。     

Identification of microRNA in the protist Trichomonas vaginalis

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. However, miRNA has never been identified experimentally in protist. Direct cloning of 438 expressed miRNA tags by microRNA serial analysis of gene expression from the parasitic protist Trichomonas vaginalis identified nine candidate miRNAs. Bioinformatics analysis of the corresponding genomic region revealed that these miRNA candidates contain a classical stem–loop–stem structure of pre-microRNAs. Analysis of the 20 nt long mature tva-miR-001 showed that it is an intergenic miRNA located at the scaffold DS113596. Tva-miR-001 was differentially expressed in the trophozoite, pseudocyst and amoeboid stages. Based on the experimental results of the present study, we provided solid evidence that protist possesses a miRNA regulating network comparable with multicellular organisms for the first time.     

Distinctive microRNA expression signatures in proton-irradiated mice

Proton particles comprise the most abundant ionizing radiation (IR) in outer space. These high energy particles are known to cause frequent double- and single-stranded DNA lesions that can lead to cancer and tumor formation. Understanding the mechanism of cellular response to proton-derived IR is vital for determining health risks to astronauts during space missions. Our understanding of the consequences of these high energy charged particles on microRNA (miRNA) regulation is still in infancy. miRNAs are non-coding, single-stranded RNAs of ~22 nucleotides that constitute a novel class of gene regulators. They regulate diverse biological processes, and each miRNA can control hundreds of gene targets. To investigate the effect of proton radiation on these master regulators, we examined the miRNA expression in selected mice organs that had been exposed to whole-body proton irradiation (2 Gy), and compared this to control mice (0 Gy exposure). RNA was isolated from three tissues (testis, brain, and liver) from treated and control mice and subjected to high-throughput small RNA sequencing. Bioinformatics analysis of small RNA sequencing data revealed dysregulation of (p < 0.05; 20 up- and 10 down-regulated) 14 mouse testis, 8 liver, and 8 brain miRNAs. The statistically significant and unique miRNA expression pattern found among three different proton-treated mouse tissues indicates a tissue-specific response to proton radiation. In addition to known miRNAs, sequencing revealed differential expression of 11 miRNAs in proton-irradiated mice that have not been previously reported in association with radiation exposure and cancer. The dysregulation of miRNAs on exposure to proton radiation suggest a possible mechanism of proton particles involvement in the onset of cell tumorgenesis. In summary, we have established that specific miRNAs are vulnerable to proton radiation, that such differential expression profile may depend upon the tissue, and that there are more miRNAs affected by proton radiation than have been previously observed.