Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using library MosSCI.
Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using library MosSCI.
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DOI：
10.1002/dvdy.24426
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发表时间：
2016-09
期刊：
影响因子：
--
通讯作者：
Ryder SP
中科院分区：
文献类型：
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作者：
Kaymak E;Farley BM;Hay SA;Li C;Ho S;Hartman DJ;Ryder SP
在线虫中，生殖系发育和早期胚胎发育依赖于母体转录的mRNAs的转录后调控。在许多情况下，3‘非编码区足以控制这些转录本的表达模式。通过3‘非编码区调控母体mRNAs需要几种RNA结合蛋白。尽管在体内对RNA结合蛋白-信使核糖核酸的相互作用进行了密集的研究，但大多数结合事件的生物学影响仍不清楚。使用单拷贝整合转基因的报告研究对于评估RNA结合蛋白及其相关mRNAs之间相互作用的功能后果是必不可少的。在这份报告中，我们提出了一种有效的方法，通过使用MosSCI转基因的文库变体来产生具有更高吞吐量的报告菌株。此外，利用RNA干扰，我们识别了控制五个不同母体mRNAs表达模式的限制性商业惯例套件。这些结果为评估体内蛋白质-RNA相互作用的功能相关性提供了一种普遍而有效的策略，并揭示了关键的RNA结合蛋白与其母体mRNA靶标之间的新的调控联系。
In C. elegans, germline development and early embryogenesis rely on post-transcriptional regulation of maternally transcribed mRNAs. In many cases, the 3′UTR is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RBPs that control the expression pattern of five different maternal mRNAs. The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets.