降解组测序技术在植物miRNA研究中的应用

目前, 利用芯片技术和miRNA测序可快速、准确地检测到物种中所含有的miRNA。随着越来越多的miRNA被发现, miRNA靶基因的确定已成为研究miRNA生物学功能的关键。传统的miRNA靶基因的寻找主要依赖生物信息学预测、AGO蛋白免疫共沉淀和荧光素酶法等。随着高通量测序技术的持续革新, 出现了一种新的miRNA靶基因的检测方法, 即降解组测序(degradome sequencing)法, 该方法拥有高通量测序技术、生物信息学分析和RACE验证三者的优势, 并已成功应用于拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)和小立碗藓(Physcomitrella patens)等模式植物miRNA靶基因的检测。基于已发表的相关文献和联川生物降解组测序平台, 该文对降解组测序技术应用于植物miRNA靶基因的研究进展及其实验原理进行了综述, 同时对运用该技术可进行的更深入研究进行了讨论。     

Functional homogeneity in microRNA target heterogeneity--a new sight into human microRNomics

MicroRNomics is a novel genomics that studies the identification, targets, biological functions, etc., of microRNAs (miRNAs) on a genomic scale. Computational target prediction algorithms are important applications in microRNomics. However, the overlaps between target sets predicted by different algorithms for one miRNA are often small. Our work is initiated to find the reasons causing "heterogeneity" and investigate whether the heterogeneous targets are homogeneous on functional levels by integrating similarity metrics. The results suggest that most human miRNAs own heterogeneous targets. The dissimilarity of thermodynamic characteristics and the different treatment of 3'-compensatory sites adopted by algorithms are the main reasons for target "heterogeneity." Meanwhile, we find most miRNA heterogeneous targets are functional homogeneity because of the common principles such as sites conservation and G:U wobble pairs in different algorithms. Our findings reveal the "functional homogeneity in miRNA target heterogeneity." The conclusions provide a perspective of microRNomics on functional levels, which introduce a new sight into human miRNA targets.     

Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

Background

In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.

Results

Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.

Conclusions

The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.     

miRSystem: An Integrated System for Characterizing Enriched Functions and Pathways of MicroRNA Targets

Background

Many prediction tools for microRNA (miRNA) targets have been developed, but inconsistent predictions were observed across multiple algorithms, which can make further analysis difficult. Moreover, the nomenclature of human miRNAs changes rapidly. To address these issues, we developed a web-based system, miRSystem, for converting queried miRNAs to the latest annotation and predicting the function of miRNA by integrating miRNA target gene prediction and function/pathway analyses.

Results

First, queried miRNA IDs were converted to the latest annotated version to prevent potential conflicts resulting from multiple aliases. Next, by combining seven algorithms and two validated databases, potential gene targets of miRNAs and their functions were predicted based on the consistency across independent algorithms and observed/expected ratios. Lastly, five pathway databases were included to characterize the enriched pathways of target genes through bootstrap approaches. Based on the enriched pathways of target genes, the functions of queried miRNAs could be predicted.

Conclusions

MiRSystem is a user-friendly tool for predicting the target genes and their associated pathways for many miRNAs simultaneously. The web server and the documentation are freely available at http://mirsystem.cgm.ntu.edu.tw/.