Department of Pediatrics, Stanford University, Stanford, CA, USA.
Department of Genetics, Stanford University, Stanford, CA, USA.
Nat Commun. 2021 Jun 7;12(1):3397. doi: 10.1038/s41467-021-23607-w.
It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.
已知 RNA 的结构决定了其生物学功能，但目前的 RNA 结构探测方法只能捕捉到部分结构信息。能够测量完整（即全长）RNA 结构将有助于研究小 RNA 的功能和调节机制，并确定功能位点的短片段。在这里，我们提出了 icSHAPE-MaP，这是一种结合体内选择性 2'-羟基酰化和突变分析来探测完整 RNA 结构的方法。我们进一步展示了基于 RNA 免疫沉淀下拉和 icSHAPE-MaP 小 RNA 结构分析相结合的人 Dicer 结合底物的 RNA 结构景观。我们发现 Dicer 底物的不同结构类别与它们的结合亲和力和切割效率相关。并且通过受 icSHAPE-MaP RNA 结构数据约束的三级结构建模，我们发现空间距离测量是 Dicer 切割位点选择的一个有影响的参数。
