A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

Identification and characterization of microRNAs in the plant parasitic root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> using deep sequencing

The root-knot nematode Meloidogyne incognita is among the most damaging plant-parasitic pests of several crops including cotton (Gossypium hirsutum) and tomato (Lycopersicon escultentum). Recently, a genome has become available for M. incognita, which greatly facilitates investigation of the interactions between M. incognita and its plant hosts at the molecular level and enables formation of hypotheses concerning development at the cellular level. MicroRNAs (miRNAs) are a class of small RNA molecules that serve as endogenous gene regulators. They regulate many biological processes including reproduction, the sequencing of morphological development, and potentially of parasitism as well. Certain miRNAs regulate fundamental metabolism pathways and stress responses in M. incognita. Since a list of miRNAs has not been generated for M. incognita, we employed a bioinformatics tool called mirDeepFinder to identify miRNAs from the small RNA database of M. incognita (GSM611102) that was generated from deep sequencing. A total of 254 conserved miRNAs belonging to 161 miRNA families were identified, as were 35 novel miRNAs belonging to 31 families. The 16 most commonly found miRNAs in order of abundance were min-miR-100a, min-miR-124, min-miR-71a, min-miR-1, min-miR-228, min-miR-92, min-miR-72, min-miR-49b, min-miR-58, min-miR-252, min-miR-lin-4, min-miR-87, min-miR-2a, min-miR-34a, min-miR-50a, and min-miR-279a. The length of the pre-miRNAs varied greatly from 50 to 197 nt, with an average of 88?±?39 nt. The average minimal folding free energy (MFE) and MFE index (MFEI) of the identified miRNAs were –30.3 Kcal/mol and 0.92, respectively, indicating that these miRNAs can readily fold into a typical hairpin secondary structure.     

Identification and analyses of miRNA genes in allotetraploid Gossypium hirsutum fiber cells based on the sequenced diploid G. raimondii genome

The plant genome possesses a large number of microRNAs（miRNAs）mainly 21-24 nucleotides in length.They play a vital role in regulation of target gene expression at various stages throughout the whole plant life cycle.Here we sequenced and analyzed～10 million non-coding RNAs（ncRNAs）derived from fiber tissue of the allotetraploid cotton（Gossypium hirsutum）1 days post-anthesis using ncRNA-seq technology.In terms of distinct reads,24 nt ncRNA is by far the dominant species,followed by 21 nt and 23 nt ncRNAs. Using ab initio prediction,we identified and characterized a total of 562 candidate miRNA gene loci on the recently assembled D₅ genome of the diploid cotton G.raimondii.Of all the 562 predicted miRNAs,22 were previously discovered in cotton species and 187 had sequence conservation and homology to homologous miRNAs of other plant species.Nucleotide bias analysis showed that the 9th and 1 st positions were significantly conserved among different types of miRNA genes.Among the 463 putative miRNA target genes,most significant up/down-regulation occurred in 10-20 days post-anthesis,indicating that miRNAs played an important role during the elongation and secondary cell wall synthesis stages of cotton fiber development.The discovery of new miRNA genes will help understand the mechanisms of miRNA generation and regulation in cotton.     

拟南芥基因组中新的microRNA预测及分析

MicroRNA（miRNA）是一类存在于动植物体内、长度为21～25nt的内源性小RNA,对生物体的转录后基因调控起着关键作用,但一些低丰度的miRNA和组织特异性miRNA往往很难发现。为了系统识别拟南芥基因组中新的非同源miRNA,首先基于已报道的拟南芥miRNA的特征,从全基因组范围中筛选出453条可能的miRNA前体;其次,为了进一步对上述miRNA前体进行筛选,利用人的miRNA前体数据构建了支持向量机模型GenomicSVM,该模型对人测试集的敏感性和特异性分别为86.3％和98．1％（30个人miRNA前体和1000个阴性miRNA前体）,对拟南芥测试集的正确率为93．6％（78个miRNA前体）;最后,利用GenomicSVM预测上述453条miRNA前体序列,得到了37条候选的新的拟南芥miRNA前体,为进一步的miRNA实验发现研究提供了指导。     

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.