Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
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DOI：
10.7554/elife.43788
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发表时间：
2019-01-17
期刊：
影响因子：
7.7
通讯作者：
Campbell, Zachary T.
中科院分区：
文献类型：
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作者：
Bhat, Vandita D.;McCann, Kathleen L.;Campbell, Zachary T.
PUF（PUmilio/FBF）RNA结合蛋白识别不同的元件。In C.在线虫中，PUF-8与8-nt基序结合并限制生殖系中的增殖。相反，FBF-2识别9-nt元件并促进有丝分裂。为了了解基序分歧与生物功能的关系，我们首先确定了PUF-8的晶体结构。这种结构的FBF-2的比较揭示了一个主要的差异，在中央重复。我们设计了一种改良的酵母3-杂交筛选，以鉴定赋予FBF-2 8-nt元件识别的突变。我们确定了几个这样的突变体，并验证其结合8-nt RNA元件的结构和生物化学。使用基因组工程，我们产生了FBF-2中具有置换的突变动物，其赋予与PUF-8元件的优先结合。突变体在很大程度上挽救了在puf-8缺失的情况下自发产生肿瘤的动物的过度增殖。这项工作突出了基序长度在生物功能规范中的关键作用。
PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.