Identification of Schistosoma mansoni microRNAs.
Identification of Schistosoma mansoni microRNAs.
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DOI：
10.1186/1471-2164-12-47
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发表时间：
2011-01-19
期刊：
影响因子：
4.4
通讯作者：
Oliveira G
中科院分区：
文献类型：
--
作者：
Simões MC;Lee J;Djikeng A;Cerqueira GC;Zerlotini A;da Silva-Pereira RA;Dalby AR;LoVerde P;El-Sayed NM;Oliveira G
MicroRNAs(MiRNAs)是一类单链RNAs，通过靶向mRNAs和引发翻译抑制或信使RNA(MRNAs)降解，在调节发育和控制基因表达方面发挥重要作用。MiRNAs广泛存在于真核生物中，到目前为止，已经通过计算和实验方法鉴定了超过14,000个miRNAs。有几个miRNAs在物种之间高度保守。在血吸虫中，全套miRNAs及其在发育过程中的表达模式仍然知之甚少。在这里，我们报告了一种基于同源性的检测策略的开发和实施，以搜索曼氏血吸虫的miRNA基因。此外，我们还报告了通过克隆和测序大小不同的RNA样本来检测miRNAs的实验结果。使用高通量管道对miRBase中所有已知的miRNAs进行同源性搜索。通过BLASTn分析，共获得6,211个成熟miRNAs作为参考序列，共返回110个曼氏沙门氏菌独特序列。报道了产生这些命中的现有成熟miRNAs，以及同源序列在曼氏血吸虫基因组中的位置。所有BLAST点击与至少95%的miRNA序列比对，导致比对长度为19-24个核苷酸。经过几个筛选步骤，使用这种方法确定了15个潜在的miRNA候选者。通过对成虫小RNA文库的测序，我们在曼氏血吸虫基因组中鉴定出211个新的miRNA候选序列。Northern印迹分析检测了30个最频繁测序的miRNAs的表达，并比较了这些miRNAs在肺期、血吸虫期和成虫期的表达水平。Northern印迹分析证实了11个新的miRNAs的表达，其中一些呈现阶段调控的表达模式。此前从日本血吸虫中鉴定出的三个miRNAs也存在于曼氏血吸虫中。提出了在曼氏葡萄球菌中存在miRNAs的证据。由于miRNA库中缺乏近亲，基于同源性的计算方法在曼氏血吸虫中检测到的miRNAs数量有限。尽管如此，这里描述的计算方法很可能适用于识别其他生物体中的前miRNA发夹。通过构建和分析一个小的RNA文库，通过分子克隆、DNA测序和表达研究相结合的方法，从曼氏链霉菌中获得了14个新的miRNAs。我们的结果大大扩展了多细胞寄生虫中已知的miRNAs的集合，并为了解这些后生动物寄生虫中miRNAs的结构和功能进化提供了基础。
MicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples. Homology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni. Evidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.
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miRNAMAP：哺乳动物基因组中microRNA基因及其靶基因的基因组图。
影响因子：
14.9
作者：
Hsu, Paul W. C.;Huang, Hsien-Da;Hsu, Sheng-Da;Lin, Li-Zen;Tsou, Ann-Ping;Tseng, Ching-Ping;Stadler, Peter F.;Washietl, Stefan;Hofacker, Ivo L.
通讯作者：
Hofacker, Ivo L.
影响因子：
4.4
作者：
Hao L;Cai P;Jiang N;Wang H;Chen Q
通讯作者：
Chen Q
影响因子：
3.7
作者：
Fahlgren N;Howell MD;Kasschau KD;Chapman EJ;Sullivan CM;Cumbie JS;Givan SA;Law TF;Grant SR;Dangl JL;Carrington JC
通讯作者：
Carrington JC
影响因子：
3.1
作者：
Baev, Vesselin;Daskalova, Evelina;Minkov, Ivan
通讯作者：
Minkov, Ivan
影响因子：
2.9
作者：
CHOMCZYNSKI, P;SACCHI, N
通讯作者：
SACCHI, N