Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins.
Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins.
复制标题
DOI：
10.1038/nchembio.2128
复制
发表时间：
2016-09
影响因子：
14.8
通讯作者：
Varani G
中科院分区：
文献类型：
--
作者：
Chen Y;Yang F;Zubovic L;Pavelitz T;Yang W;Godin K;Walker M;Zheng S;Macchi P;Varani G
RNA识别基序(RRM)是真核生物中最大的RNA结合蛋白家族。具有新的特异性的工程RRMS将提供有价值的工具和对我们对特异性的理解的严格测试。我们已经成功地使用Rational方法成功地重新设计了RRM的特异性，并证明了细胞中活性的重新定位。我们设计了人RBFox蛋白的保守RRM，以高亲和力特异性地与miR-21前体的末端环结合，并抑制其被DROSHA和DICER加工。我们用设计的RRM取代了它的PAZ结构域，从而进一步工程了贾迪亚·迪格尔。这种重新编程的酶在体外特异性地降解前miR-21，并抑制细胞中成熟的miR-21水平，导致PDCD4表达增加，显著降低癌细胞的生存能力。这些结果证明了设计RRMS序列特异性的可行性，并将这个无处不在的平台用于不同的生物应用。
The RNA Recognition Motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with new specificity would provide valuable tools and an exacting test of our understanding of specificity. We have achieved the first successful re-design of the specificity of an RRM using rational methods and demonstrated re-targeting of activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of miR-21 precursor with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.