Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.
Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.
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DOI：
10.1261/rna.2286706
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发表时间：
2006-04
期刊：
影响因子：
4.5
通讯作者：
K. Riley;L. Cassiday;Akash Kumar;L. J. Maher
中科院分区：
文献类型：
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作者：
K. Riley;L. Cassiday;Akash Kumar;L. J. Maher
p53肿瘤抑制蛋白是一种同四聚体转录因子，其基因在近一半的人类癌症中发生突变。在使用酵母三杂交系统的RNA/蛋白相互作用的不相关筛选中，我们无意中检测到p53与几种不同RNA的相互作用。一篇文献综述揭示了先前关于p53和RNA之间序列特异性和非特异性相互作用的报道。使用酵母三杂交选择来确定p53的首选RNA伴侣，我们未能确定p53结合所需的初级RNA序列或明显的二级结构。在酵母中，阳离子p53 c端被证明是RNA结合所必需的。我们发现，虽然p53在酵母三杂交实验中强烈区分某些rna，但相同的rna在体外被p53平等地结合。我们进一步表明，酵母中的p53 rna结合偏好几乎完全反映了HIV-1核衣壳（NC）蛋白的重组四聚体形式，该蛋白被认为是一种序列非特异性rna结合蛋白。但不能排除p53特异性结合RNA的可能性，因为p53与HIV-1 NC在RNA结合偏好上存在一定差异。我们得出结论：(1)p53在体内与RNA结合，(2)在酵母细胞核中，p53与RNA的结合在很大程度上是非序列特异性的，(3)不能排除p53与一些结构特异性RNA的结合，(4)在解释酵母三杂交系统中的RNA筛选结果时需要谨慎，因为RNA折叠和显示的序列依赖性差异可以伪装成蛋白质识别的序列依赖性差异。
The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.