Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

中文翻译：

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用于亲和纯化和RNA-蛋白质相互作用鉴定的酶法RNA生物素化。

在整个细胞生命周期中，RNA转录物均与蛋白质结合，在RNA代谢，运输和功能中起着至关重要的作用。尽管这些相互作用很重要，但在哺乳动物细胞中鉴定与目标RNA相互作用的蛋白质仍是RNA生物学的主要挑战。利用利用大肠杆菌tRNA鸟嘌呤转糖基酶和非天然核碱基底物对目标RNA进行位点特异性和共价标记的能力，我们建立了RNA-蛋白相互作用的鉴定和哺乳动物系统中细胞RNA的选择性富集。我们通过质谱鉴定7SK snRNA的已知结合伴侣，证明了这种方法的实用性。通过长的非编码RNA HOTAIR的最小4个核苷酸突变，酶促生物素化可以鉴定MCF7乳腺癌细胞中假定的HOTAIR结合伴侣，从而提示致癌功能的新潜在途径。此外，使用RNA测序和qPCR，我们建立了一种工程化的酶变体，可以实现针对人类转录组的高水平标记选择性，从而可以直接从哺乳动物细胞裂解物中富集145倍的细胞RNA。这种方法的灵活性和广度表明，该系统可常规应用于RNA的功能表征，从而大大扩展了可用于研究哺乳动物RNA生物学的工具箱。我们建立了一种工程化的酶变体，可以实现对人类转录组的高水平标记选择性，从而可以直接从哺乳动物细胞裂解物中富集145倍的细胞RNA。这种方法的灵活性和广度表明，该系统可常规应用于RNA的功能表征，从而大大扩展了可用于研究哺乳动物RNA生物学的工具箱。我们建立了一种工程化的酶变体，可以实现对人类转录组的高水平标记选择性，从而可以直接从哺乳动物细胞裂解物中富集145倍的细胞RNA。这种方法的灵活性和广度表明，该系统可常规应用于RNA的功能表征，从而大大扩展了可用于研究哺乳动物RNA生物学的工具箱。