Modified nucleotide cofactors and the interaction of the RNAome with the proteome

描述(由申请人提供)：我们有初步证据表明，siRNA和miRNAs大小的小RNA与辅酶A和bi-MGD等修饰核苷酸偶联。这些修饰将允许小RNA与蛋白质组共价结合，在这些蛋白质上构成核酸条形码。我们建议使用强大的核酸杂交和选择技术来纯化与RNA共价连接的蛋白质，并使用质谱学来鉴定这些连接的蛋白质。利用这些蛋白质的RNAi，我们将调查小RNA功能的缺陷，从而发现这些蛋白质参与小RNA的功能。因为所有的修饰都被预测发生在小RNA的5‘端，所以大多数这些修饰的RNA将被系统地遗漏，因为大多数研究需要在RNA上使用5’OH或5‘磷酸来进行克隆。 公共卫生相关性：我们的初步数据表明，修饰的核苷酸参与了小RNA的功能。这项研究的意义是巨大的：如果这样的小RNA是RNAi和miRNA功能的活性成分，因为它与蛋白质组的结合有关，那么小RNA领域可能系统地错过了小RNA的一个主要世界。以同样的方式，由于不从生物学上看22个核苷酸，由于不能检测到带有5‘修饰的小RNA，小RNA领域可能会有另一种暗物质情况。我们会补救这一点。

DESCRIPTION (provided by applicant): We have preliminary evidence that small RNAs of the size of siRNA and miRNAs are coupled to modified nucleotides such as coenzyme A and bis-MGD. These modifications would allow small RNAs to covalently engage with the proteome to constitute nucleic acid bar codes on these proteins. We propose to use powerful nucleic acid hybridization and selection techniques to purify the proteins covalently linked to RNA and to use mass spectroscopy to identify those linked proteins. Using RNAi of these proteins we will survey for defects in small RNA function and thus an involvement of these proteins in the functions of small RNAs. Because all of the modifications are predicted to occur at the 5' end of small RNAs, most of these modified RNAs will have been systematically missed by surveys most of which demands 5' OH or 5' phosphates on the RNAs for cloning. PUBLIC HEALTH RELEVANCE: Our preliminary data points to the involvement of modified nucleotides in small RNA function. The implications of this research are enormous: if such small RNAs are the active components of RNAi and miRNA function, as it pertains to the engagement with the proteome, then the field of small RNAs may have systematically missed a major world of small RNAs. In the same way that by not looking at 22 nt for biology, by not being able to detect small RNAs with 5' modifications, the small RNA field may have another dark matter situation. We will remedy this.