Prediction of Conserved Precursors of miRNAs and Their Mature Forms by Integrating Position-Specific Structural Features

MicroRNA (miRNA) precursor hairpins have a unique secondary structure, nucleotide length, and nucleotide content that are in most cases evolutionarily conserved. The aim of this study was to utilize position-specific features of miRNA hairpins to improve their identification. To this end, we defined the evolutionary and structurally conserved features in each position of miRNA hairpins with heuristically derived values, which were successfully integrated using a probabilistic framework. Our method, miRRim2, can not only accurately detect miRNA hairpins, but infer the location of a mature miRNA sequence. To evaluate the accuracy of miRRim2, we designed a cross validation test in which the whole human genome was used for evaluation. miRRim2 could more accurately detect miRNA hairpins than the other computational predictions that had been performed on the human genome, and detect the position of the 5'-end of mature miRNAs with sensitivity and positive predictive value (PPV) above 0.4. To further evaluate miRRim2 on independent data, we applied it to the Ciona intestinalis genome. Our method detected 47 known miRNA hairpins among top 115 candidates, and pinpointed the 5'-end of mature miRNAs with sensitivity and PPV about 0.4. When our results were compared with deep-sequencing reads of small RNA libraries from Ciona intestinalis cells, we found several candidates in which the predicted mature miRNAs were in good accordance with deep-sequencing results.     

Discovery of novel microRNAs in rat kidney using next generation sequencing and microarray validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.     

Identification of conserved microRNAs and their targets from Solanum lycopersicum Mill

MicroRNAs (miRNAs) are a class of highly conserved endogenous non-coding small RNAs crossing kingdoms of organisms. By searching known miRNAs identified from plant species against tomato nucleotide sequences, 13 pre-miRNAs of the nine mature miRNAs were found, amongst, six had been cloned. To confirm our prediction, a miRNA-detecting microarray was designed with probes complementary to all non-redundant mature plant miRNAs documented to date. After hybridizing with small RNAs extracted from tomato leaf tissue, 78 highly expressed mature miRNAs were detected, including all the miRNAs predicted above. Conformation of some miRNAs expression by Northern hybridization indicated that they were highly accumulated not only in leaf tissues but also in roots and stems. Additional BLAST searches with newly recognized miRNAs against tomato mRNAs from NCBI yielded 23 potential targets mainly associated with the phase change from vegetative to generative growth, with flower development and with responding to plant hormone and virus stress.     

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.     

Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus.

RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.     

红花microRNAs靶基因的生物信息学预测

miRNA是一类动、植物细胞中负责转录后调控的非编码RNA,植物的miRNA通常来源于具有茎环结构的单链RNA前体上,加工成熟后同ARGONAUTE转变成蛋白复合体结构,通过结合并沉默蛋白合成模板的方式关闭完整的基因表达网络。由于miRNA存在组织表达的特异性,且miRNA在植物中的表达高度保守。通过高通量测序获得的红花(Carthamus tinctorious L.)miRNA序列信息与红花转录EST数据库比对,通过靶基因预测筛选,对属于104个家族的173个红花miRNA进行预测,其中得到109个miRNAs对应调控的385个红花靶基因。并且通过Nr基因注释表明多数红花miRNA的靶基因编码包括调控细胞生长发育、信号转导及新陈代谢等相关的功能蛋白。     

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.     

Computational identification of miRNAs in medicinal plant Senecio vulgaris (Groundsel)

RNAs Interference plays a very important role in gene silencing. In vitro identification of miRNAs is a slow process as it is difficult to isolate them. Nucleotide sequences of miRNAs are highly conserved among the plants and, this form the key feature behind the identification of miRNAs in plant species by homology alignment. In silico identification of miRNAs from EST database is emerging as a novel, faster and reliable approach. Here EST sequences of Senecio vulgaris (Groundsel) were searched against known miRNA sequences by using BLASTN tool. A total of 10 miRNAs were identified from 1956 EST sequences and 115 GSS sequences. The most stable miRNA identified is svu-mir-1. This approach will accelerate advance research in regulation of gene expression in Groundsel by interfering RNAs.