Identification and characteristics of microRNAs from Bombyx mori.
Identification and characteristics of microRNAs from Bombyx mori.
复制标题
家蚕 microRNA 的鉴定和特征。
DOI：
10.1186/1471-2164-9-248
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发表时间：
2008-05-28
期刊：
影响因子：
4.4
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
He PA;Nie Z;Chen J;Chen J;Lv Z;Sheng Q;Zhou S;Gao X;Kong L;Wu X;Jin Y;Zhang Y
MicroRNAs(MiRNAs)是一种小的RNA分子，通过靶向信使RNAs(MRNAs)调节基因的表达，并导致mRNA切割或翻译阻断。在已鉴定的355个节肢动物miRNAs中，只有21个是通过计算预测的家蚕miRNAs；其中只有let-7被Northern blotting证实。将基于序列同源性搜索的计算方法与基于微阵列分析和Northern blotting的实验鉴定相结合，我们在家蚕中鉴定出46个miRNAs，另外21个可信的miRNAs，以及一个新的小RNA。后者是用已知的miRNA Aga-miR-100作为探针鉴定的，BMO-miR-100类似物通过基因芯片和Northern blotting检测到，但其前体序列没有折叠成发夹结构。在这些已鉴定的miRNAs中，我们发现了12对miRNAs和miRNA*S。Northern blotting表明，一些家蚕miRNA基因只在特定的时期表达，表明家蚕miRNA基因(如BMO-miR-277)具有发育调控的表达模式。我们在家蚕基因组中发现了两个miRNA基因簇。在基因簇bmo-miR-2a-1/bmo-miR-2a-1*/bmo-miR-2a-2/bmo-miR-2b/bmo-miR-13a*/bmo-miR-13b，中发现的bmo-miR-2b编码了一个新发现的mir-2家族成员。此外，我们发现甲基化可以提高用于Northern印迹检测miRNA的DNA探针的灵敏度。功能分析表明，根据功能保守，11个miRNAs可能调控已知的25个果蝇miRNA靶基因中的13个家蚕同源基因。我们预测了家蚕B.mori基因1671个3‘非编码区的结合位点；预测了547个靶基因，包括986个靶点。在这些靶点中，有338个与43个miRNAs的种子区具有完美的碱基配对。从预测的基因中，61个基因，每个基因都有多个预测的靶点，应该被认为是未来功能研究的优秀候选基因。对预测的miRNA靶标的生物学分类表明，预测基因的“结合”、“催化活性”和“生理过程”被过度代表。结合计算机预测和微阵列分析，我们鉴定了46个家蚕白僵菌miRNAs，其中13个为S；我们发现了一个新的小RNA和21个可能的家蚕白僵菌miRNAs，这些基因不能在现有的家蚕白僵菌基因组中定位，但可以被微阵列检测到。根据功能保守区和结合位点分别预测了13个和547个目的基因。鉴定家蚕中的miRNAs，特别是那些受发育调控的miRNAs，为后续的功能研究提供了基础。
MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by targeting messenger RNAs (mRNAs) and causing mRNA cleavage or translation blockage. Of the 355 Arthropod miRNAs that have been identified, only 21 are B. mori miRNAs that were predicted computationally; of these, only let-7 has been confirmed by Northern blotting. Combining a computational method based on sequence homology searches with experimental identification based on microarray assays and Northern blotting, we identified 46 miRNAs, an additional 21 plausible miRNAs, and a novel small RNA in B. mori. The latter, bmo-miR-100-like, was identified using the known miRNA aga-miR-100 as a probe; bmo-miR-100-like was detected by microarray assay and Northern blotting, but its precursor sequences did not fold into a hairpin structure. Among these identified miRNAs, we found 12 pairs of miRNAs and miRNA*s. Northern blotting revealed that some B. mori miRNA genes were expressed only during specific stages, indicating that B. mori miRNA genes (e.g., bmo-miR-277) have developmentally regulated patterns of expression. We identified two miRNA gene clusters in the B. mori genome. bmo-miR-2b, which is found in the gene cluster bmo-miR-2a-1/bmo-miR-2a-1*/bmo-miR-2a-2/bmo-miR-2b/bmo-miR-13a*/bmo-miR-13b, encodes a newly identified member of the mir-2 family. Moreover, we found that methylation can increase the sensitivity of a DNA probe used to detect a miRNA by Northern blotting. Functional analysis revealed that 11 miRNAs may regulate 13 B. mori orthologs of the 25 known Drosophila miRNA-targeted genes according to the functional conservation. We predicted the binding sites on the 1671 3'UTR of B. mori genes; 547 targeted genes, including 986 target sites, were predicted. Of these target sites, 338 had perfect base pairing to the seed region of 43 miRNAs. From the predicted genes, 61 genes, each of them with multiple predicted target sites, should be considered excellent candidates for future functional studies. Biological classification of predicted miRNA targets showed that "binding", "catalytic activity" and "physiological process" were over-represented for the predicted genes. Combining computational predictions with microarray assays, we identified 46 B. mori miRNAs, 13 of which were miRNA*s. We identified a novel small RNA and 21 plausible B. mori miRNAs that could not be located in the available B. mori genome, but which could be detected by microarray. Thirteen and 547 target genes were predicted according to the functional conservation and binding sites, respectively. Identification of miRNAs in B. mori, particularly those that are developmentally regulated, provides a foundation for subsequent functional studies.
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