水稻MicroRNA的预测及实验验证

根据已报道水稻pre-miRNA的序列与结构信息,利用支持向量机（support vector machine, SVM）方法在miRNA前体上预测成熟区,产生一个模型——mature-SVM.它预测水稻成熟区的敏感性和特异性分别为86.7% 和100%;然后,用这个模型对从水稻基因组中筛选出的46.501条pre-miRNA进行成熟链预测,此外再根据miRNA的作用原理用blast程序所进一步的筛选,得到了127条pre-miRNA及成熟miRNA;除去其中已知的21条,最后得到106条候选的新的水稻miRNA. 从中随机挑取10条进行Northern验证,结果有4条miRNA得到确认.     

MiRANN: a reliable approach for improved classification of precursor microRNA using Artificial Neural Network model

MicroRNA (miRNA) is a special class of short noncoding RNA that serves pivotal function of regulating gene expression. The computational prediction of new miRNA candidates involves various methods such as learning methods and methods using expression data. This article has proposed a reliable model - miRANN which is a supervised machine learning approach. MiRANN used known pre-miRNAs as positive set and a novel negative set from human CDS regions. The number of known miRNAs is now huge and diversified that could cover almost all characteristics of unknown miRNAs which increases the quality of the result (99.9% accuracy, 99.8% sensitivity, 100% specificity) and provides a more reliable prediction. MiRANN performs better than other state-of-the-art approaches and declares to be the most potential tool to predict novel miRNAs. We have also tested our result using a previous negative set. MiRANN, opens new ground using ANN for predicting pre-miRNAs with a promise of better performance.     

A whole-genome RNAi Screen for C. elegans miRNA pathway genes

BACKGROUND: miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs downregulate target gene expression. An exploration of which protein cofactors are necessary for a miRNA to downregulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes. RESULTS: A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the downregulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes. CONCLUSIONS: The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or for recognizing the miRNA base-paired to the target mRNA to downregulate translation.     

Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus

We used an in silico approach to predict microRNAs (miRNAs) genome-wide in the brown alga Ectocarpus siliculosus. As brown algae are phylogenetically distant from both animals and land plants, our approach relied on features shared by all known organisms, excluding sequence conservation, genome localization and pattern of base-pairing with the target. We predicted between 500 and 1500 miRNAs candidates, depending on the values of the energetic parameters used to filter the potential precursors. Using quantitative polymerase chain reaction assays, we confirmed the existence of 22 miRNAs among 72 candidates tested, and of 8 predicted precursors. In addition, we compared the expression of miRNAs and their precursors in two life cycle states (sporophyte, gametophyte) and under salt stress. Several miRNA precursors, Argonaute and DICER messenger RNAs were differentially expressed in these conditions. Finally, we analyzed the gene organization and the target functions of the predicted candidates. This showed that E. siliculosus miRNA genes are, like plant miRNA genes, rarely clustered and, like animal miRNA genes, often located in introns. Among the predicted targets, several widely conserved functional domains are significantly overrepresented, like kinesin, nucleotide-binding/APAF-1, R proteins and CED-4 (NB-ARC) and tetratricopeptide repeats. The combination of computational and experimental approaches thus emphasizes the originality of molecular and cellular processes in brown algae.     

Phylogenetic shadowing and computational identification of human microRNA genes

We sequenced 122 miRNAs in 10 primate species to reveal conservation characteristics of miRNA genes. Strong conservation is observed in stems of miRNA hairpins and increased variation in loop sequences. Interestingly, a striking drop in conservation was found for sequences immediately flanking the miRNA hairpins. This characteristic profile was employed to predict novel miRNAs using cross-species comparisons. Nine hundred and seventy-six candidate miRNAs were identified by scanning whole-genome human/mouse and human/rat alignments. Most of the novel candidates are conserved also in other vertebrates (dog, cow, chicken, opossum, zebrafish). Northern blot analysis confirmed the expression of mature miRNAs for 16 out of 69 representative candidates. Additional support for the expression of 179 novel candidates can be found in public databases, their presence in gene clusters, and literature that appeared after these predictions were made. Taken together, these results suggest the presence of significantly higher numbers of miRNAs in the human genome than previously estimated.     

Caenorhabditis elegans and the study of gene function in parasites.

The free-living nematode Caenorhabditis elegans is a tractable experimental model system for the study of both vertebrate and invertebrate biology. Its most significant advantages are its simplicity, both in anatomy and in genomic organization, and the elaborate methods that have been developed to attribute function to previously uncharacterized genes. Importantly, > 40% of parasitic nematode genes exhibit high levels of homology to genes within the C. elegans genome. Studying such genes using the C. elegans model should yield new insights into key molecules and their possible implications in parasite survival, leading to the discovery of new drug targets and vaccine candidates.     

Oncomirs: the potential role of non-coding microRNAs in understanding cancer

MicroRNAs (miRNAs) are members of a family of non-coding RNAs of 8-24 nucleotide RNA molecules that regulate target mRNAs. The first miRNAs, lin-4 and let-7, were first discovered in the year 1993 by Ambros, Ruvkun, and co-workers while studying development in Caenorhabditis elegans. miRNAs can play vital functions form C. elegans to higher vertebrates by typical Watson-Crick base pairing to specific mRNAs to regulate the expression of a specific gene. It has been well established that multicellular eukaryotes utilize miRNAs to regulate many biological processes such as embryonic development, proliferation, differentiation, and cell death. Recent studies have shown that miRNAs may provide new insight in cancer research. A recent study demonstrated that more than 50% of miRNA genes are located in fragile sites and cancer-associated genomic regions, suggesting that miRNAs may play a more important role in the pathogenesis of human cancers. Exploiting the emerging knowledge of miRNAs for the development of new human therapeutic applications will be important. Recent studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer. In this review, we focus on how miRNAs regulate tumorigenesis by acting as oncogenes and anti-oncogenes in higher eukaryotes.     

Identification of novel microRNAs in rice (Oryza sativa) based on the cleavage signals in precursors

MicroRNAs (miRNAs) are crucial regulators of gene expression in plants and a growing number of novel miRNA genes have been cloned in rice in recent years. However, there is no evidence that all miRNAs have been discovered, especially for those low expression ones which are difficult to be found by conventional methods. By taking advantage of the finding that DCL1-mediated cleavage signals for the processing of the miRNA precursors could be used as the clues for novel miRNAs’ discovery, a genome-wide search for rice miRNA candidates was carried out. As a result, 51 previously validated miRNAs and 24 novel miRNA candidates were discovered. After target prediction and degradome sequencing data-based validation, coupled with reverse approach retest, 10 miRNA candidate–mRNA target pairs were further identified, providing a basis for in-depth functional analysis of these miRNA candidates. Besides, some isomiRs found in this study showed more likely to be the real miRNAs. We also found an exceptional example which did not obey the rule that 22-nt miRNAs have the ability to trigger the phased siRNAs production from the cleaved targets.