A whole-genome RNAi screen for C-elegans miRNA pathway genes
A whole-genome RNAi screen for C-elegans miRNA pathway genes
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DOI：
10.1016/j.cub.2007.10.058
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发表时间：
2007-12-04
期刊：
影响因子：
9.2
通讯作者：
Ruvkun, Gary
中科院分区：
文献类型：
--
作者：
Parry, Devin H.;Xu, Jinling;Ruvkun, Gary
背景：miRNAs是一类丰富的内源性小分子调控RNA。尽管现在认识到miRNA参与了广泛的生物学过程，但对于miRNA下调靶基因表达的实际机制知之甚少。探索哪些蛋白辅因子是miRNA下调靶基因所必需的，应该更全面地揭示miRNA加工、运输和调节其靶基因的分子机制。以线虫miRNA基因let-7为致敏遗传背景进行全基因组RNAi筛选，共筛选出213个候选miRNA通路基因。通过遗传测试验证了具有最强表型的61个候选者中的约2/3，所述遗传测试检查let-7表型对已知在let-7途径中起作用的靶基因的依赖性。let-7 miRNA产生的生化测试将几乎所有这些新的miRNA途径基因的功能置于let-7表达和加工的下游。通过监测lin-14 mRNA的蛋白产物的下调，这是lin-4 miRNA.Conclusions的目标，我们已经确定了19个一般的miRNA通路基因：213个候选的miRNA通路基因可以在步骤，产生和交通的miRNA或在下游步骤，检测miRNA：：mRNA双链体调节mRNA的翻译。这19个经过验证的一般miRNA途径基因是可能定义蛋白质辅因子的基因的良好候选者，所述蛋白质辅因子用于分选或靶向miRNA：：mRNA双链体，或用于识别与靶mRNA碱基配对的miRNA以下调翻译。
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.