Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.     

An Exploration of Evolution,Maturation, Expression and Function Relationships in Mir-23～27～24 Cluster

The study aims to explore the potential relationships of evolution, maturation, expression and function between homologous/clustered miRNAs. mir-23∼27∼24 gene cluster, including the two gene clusters (mir-23a and mir-23b) and the three miRNA gene families (mir-23, mir-27 and mir-24), was typically selected as an example. These related miRNAs show similar evolutionary patterns and various expression patterns. Most of them show consistent isomiR expression pattern, and the “switching” phenomenon can be found between different abundant isomiR species. These findings suggest that these sequence or location related miRNAs show the similar miRNA processing and maturation processes, and the robust selection of the most dominant isomiR exists in specific tissues. Functional analysis show that these miRNAs show similar distributions of enriched gene categories, suggesting the close functional prelateships via direct or indirect coordinate regulation in biological processes. The study reveals the close evolutionary, expression and functional relationships between related homologous/clustered miRNAs, which will further enrich miRNA studies and understand direct or indirect interactions between miRNAs.     

Genome-wide analysis of aberrantly expressed circulating miRNAs in patients with coal workers’ pneumoconiosis

Emerging evidence indicates that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, have important roles in multiple biological processes. To determine the potential contribution of miRNAs to coal workers’ pneumoconiosis (CWP), we comprehensively surveyed and identified differentially expressed miRNA profiles in patients with CWP by small RNA sequencing and analysis. Mixed serum samples from the different stages of CWP and the control samples were subjected to deep sequencing by applying next-generation sequencing technology. Samples at different disease stages exhibited inconsistent miRNA expression profiles and differentially expressed miRNA profiles. Generally, these miRNAs were dynamically expressed across the different disease stages and showed various relative expression levels. Some miRNAs (such as miR-18a*, 149, 222 and 671-3p) were consistently up-regulated or down-regulated in the different stages of CWP samples. Most of the aberrantly expressed miRNAs showed a down-regulation trend. Differentially expressed miRNAs were also subjected to pairwise comparison between the different stages. Some miRNAs showed significant inconsistent expression trends across the three stages, although they were not significantly dysregulated based on the control sample. Furthermore, a series of special miRNAs organized into miRNA gene clusters and gene families were also surveyed for aberrant expression (such as mir-200 gene family and mir-222 gene cluster). According to experimentally validated target mRNAs of the aberrantly and abundantly expressed miRNAs, functional enrichment analysis suggests that these miRNAs play important roles in various biological processes, including lung tumorigenesis. In summary, we demonstrated that aberrantly expressed circulating miRNAs showed dynamic expression patterns across diseased samples, which suggests that these miRNAs may have critical roles in the occurrence and development of CWP. In addition, some significantly dysregulated miRNAs may be potential non-invasive diagnosis biomarkers based on further study.     

Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development

Background

Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous ∼21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo.

Methods and Findings

We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides—not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes.

Conclusions

These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.     

固始鸡1日龄和36周龄下丘脑高丰度差异性MiRNA的鉴定及功能预测分析

为鉴定鸡下丘脑发育相关特异性表达miRNA,基于固始鸡1日龄和36周龄下丘脑小RNA的Solexa测序数据,共鉴定到266种2个发育阶段共表达的miRNA,其中157种miRNA的表达水平被显著下调,22种被显著上调.聚类分析显示,鸡下丘脑高丰度差异性miRNA主要集中于let-7、mir-181、mir-30、mir-99、mir-1和mir-17等基因家族.另外,预测了10种高丰度差异性miRNA的靶基因,并进行了相应的GO分析和KEGG通路分析.结果显示,预测靶基因在发育过程、代谢过程、细胞过程和生物学过程调节等4个生物学过程以及细胞周期、粘着斑、TGF-beta信号通路和MAPK信号通路等通路中显著富集.研究结果为进一步揭示miRNA调控鸡下丘脑发育的分子机制提供了有益线索.     

SAGE detects microRNA precursors

Background

MicroRNAs (miRNAs) have been shown to play important roles in regulating gene expression. Since miRNAs are often evolutionarily conserved and their precursors can be folded into stem-loop hairpins, many miRNAs have been predicted. Yet experimental confirmation is difficult since miRNA expression is often specific to particular tissues and developmental stages.

Results

Analysis of 29 human and 230 mouse longSAGE libraries revealed the expression of 22 known and 10 predicted mammalian miRNAs. Most were detected in embryonic tissues. Four SAGE tags detected in human embryonic stem cells specifically match a cluster of four human miRNAs (mir-302a, b, c&d) known to be expressed in embryonic stem cells. LongSAGE data also suggest the existence of a mouse homolog of human and rat mir-493.

Conclusion

The observation that some orphan longSAGE tags uniquely match miRNA precursors provides information about the expression of some known and predicted miRNAs.

     

MicroRNA-181b and microRNA-9 mediate arsenic-induced angiogenesis via NRP1

Environmental exposure to inorganic arsenic compounds has been reported to have serious health effects on humans. Recent studies reported that arsenic targets endothelial cells lining blood vessels, and endothelial cell activation or dysfunction, may underlie the pathogenesis of arsenic-induced diseases and developmental toxicity. It has been reported that microRNAs (miRNAs) may act as an angiogenic switch by regulating related genes. The present study was designed to test the hypothesis that arsenite-regulated miRNAs play pivotal roles in arsenic-induced toxicity. Fertilized eggs were injected via the yolk sac with 100 nM sodium arsenite at Hamburger-Hamilton (HH) stages 6, 9, and 12, and harvested at HH stage 18. To identify the individual miRNAs and mRNAs that may regulate the genetic network, the expression profiles of chick embryos were analyzed by microarray analysis. Microarray analyses revealed that the expression of a set of miRNAs changed after arsenite administration, especially miRNA-9, 181b, 124, 10b, and 125b, which exhibited a massive decrease in expression. Integrative analyses of the microarray data revealed that several miRNAs, including miR-9 and miR-181b, might target several key genes involved in arsenic-induced developmental toxicity. A luciferase reporter assay confirmed neuropilin-1 (Nrp1) as a target of mir-9 and mir-181b. Data from the transwell migration assay and the tube-formation assay indicated that miR-9 and mir-181b inhibited the arsenic-induced EA.hy926 cell migration and tube formation by targeting NRP1. Our study demonstrates that the environmental toxin, sodium arsenite, induced angiogenesis by altering the expression of miRNAs and their cognate mRNA targets.     

Comparative genomic analysis reveals evolutionary characteristics and patterns of microRNA clusters in vertebrates

MicroRNAs (miRNAs) are a class of small non-coding RNAs that can play important regulatory roles in many important biological processes. Although clustering patterns of miRNA clusters have been uncovered in animals, the origin and evolution of miRNA clusters in vertebrates are still poorly understood. Here, we performed comparative genomic analyses to construct 51 sets of orthologous miRNA clusters (SOMCs) across seven test vertebrate species, a collection of miRNA clusters from two or more species that are likely to have evolved from a common ancestral miRNA cluster, and used these to systematically examine the evolutionary characteristics and patterns of miRNA clusters in vertebrates. We found that miRNA clusters are continuously generated, and most of them tend to be conserved and maintained in vertebrate genomes, although some adaptive gains and losses of miRNA cluster have occurred during evolution. Furthermore, miRNA clusters appeared relatively early in the evolutionary history might suffer from more complicated adaptive gain-and-loss than those young miRNA clusters. Detailed analysis showed that genomic duplication events of ancestral miRNAs or miRNA clusters are likely to be major driving force and apparently contribute to origin and evolution of miRNA clusters. Comparison of conserved with lineage-specific miRNA clusters revealed that the contribution of duplication events for the formation of miRNA cluster appears to be more important for conserved miRNA clusters than lineage-specific. Our study provides novel insights for further exploring the origins and evolution of miRNA clusters in vertebrates at a genome scale.     

MetaMirClust: Discovery of miRNA cluster patterns using a data-mining approach

Recent genome-wide surveys on ncRNA have revealed that a substantial fraction of miRNA genes is likely to form clusters. However, the evolutionary and biological function implications of clustered miRNAs are still elusive. After identifying clustered miRNA genes under different maximum inter-miRNA distances (MIDs), this study intended to reveal evolution conservation patterns among these clustered miRNA genes in metazoan species using a computation algorithm. As examples, a total of 15-35% of known and predicted miRNA genes in nine selected species constitute clusters under the MIDs ranging from 1kb to 50kb. Intriguingly, 33 out of 37 metazoan miRNA clusters in 56 metazoan genomes are co-conserved with their up/down-stream adjacent protein-coding genes. Meanwhile, a co-expression pattern of miR-1 and miR-133a in the mir-133-1 cluster has been experimentally demonstrated. Therefore, the MetaMirClust database provides a useful bioinformatic resource for biologists to facilitate the advanced interrogations on the composition of miRNA clusters and their evolution patterns.     

Identification of novel chicken microRNAs and analysis of their genomic organization

MicroRNAs (miRNAs) represent a family of small noncoding RNAs with important regulatory roles in diverse biological processes ranging from cell differentiation to organism development. In chickens, the full set of miRNAs and the expression patterns of miRNAs during development are still poorly understood when compared to the other vertebrates. In this study, we identified 29 novel miRNAs and 140 potential miRNA loci in the chicken genome by combining the experimental and computational analyses. Detailed expression patterns of 49 miRNAs were first characterized by Northern blotting and indicated the cooperativity of the miRNA expression with their function in embryogenesis and organogenesis. Twenty-seven miRNA clusters were systematically evaluated in the chicken genome and diverse expression patterns for closely linked miRNAs were observed. Our results significantly expand the set of known miRNAs in the chicken and provide the basis for understanding the structural and functional evolution of miRNA genes in vertebrates.