Identification of proteins and miRNAs that specifically bind an mRNA in vivo
Identification of proteins and miRNAs that specifically bind an mRNA in vivo
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DOI：
10.1038/s41467-019-12050-7
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发表时间：
2019-09
影响因子：
16.6
通讯作者：
Kathrin Theil;Koshi Imami;N. Rajewsky
中科院分区：
文献类型：
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作者：
Kathrin Theil;Koshi Imami;N. Rajewsky
理解mRNA的调控需要了解其调控因子。然而，用于可靠的从头鉴定与特定RNA结合的蛋白质的方法是稀缺的，并且迄今为止仅成功地应用于细胞培养物中丰富的非编码RNA。在这里，我们提出了vIPR，RNA-蛋白质交联，RNA下拉，鸟枪蛋白质组学方法，以确定蛋白质结合到选定的mRNA在C。优雅的。将vIPR应用于生殖系特异性转录物gld-1导致已知和新型相互作用物的富集。通过比较在ld-1和lin-41 pulldown上的富集，我们证明vIPR回收了常见的和特异性的RNA结合蛋白，并且我们验证了DAZ-1作为一种特异性的gld-1调节剂。最后，结合vIPR和小RNA测序，我们恢复了已知的和生物学上重要的转录特异性miRNA相互作用，我们确定miR-84为gld-1转录的特异性相互作用因子。我们设想，vIPR将提供一个平台，研究RNA在不同的生物系统中的体内调控。
Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs inC. elegans. Applying vIPR to the germline-specific transcriptgld-1led to enrichment of known and novel interactors. By comparing enrichment upongld-1andlin-41pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specificgld-1regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identifymiR-84as a specific interactor of thegld-1transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.