Brown V, Small K, Lakkis L, Feng Y, Gunter C, Wilkinson K D, Warren S T
Howard Hughes Medical Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 1998 Jun 19;273(25):15521-7. doi: 10.1074/jbc.273.25.15521.
Fragile X syndrome is caused by the transcriptional silencing of the FMR1 gene due to a trinucleotide repeat expansion. The encoded protein, Fmrp, has been found to be a nucleocytoplasmic RNA-binding protein containing both KH domains and RGG boxes that associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remained uncertain whether the selective RNA binding observed was an intrinsic property of Fmrp or required an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibited selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp bound to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrated RNA binding, in contrast to previous suggestions. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands.
脆性X综合征是由FMR1基因因三核苷酸重复序列扩增导致转录沉默引起的。已发现编码的蛋白质Fmrp是一种核质RNA结合蛋白，含有KH结构域和RGG框，作为核糖核蛋白颗粒与多核糖体结合。此前已通过体外翻译的Fmrp证实了RNA结合；然而，观察到的选择性RNA结合是Fmrp的固有特性还是需要相关蛋白质仍不确定。在这里，杆状病毒表达并经亲和纯化的带有FLAG标签的小鼠Fmrp被证明可直接与核糖核苷酸同聚物和人脑mRNA结合。FLAG-Fmrp对结合poly(G) > poly(U) >> poly(C)或poly(A)表现出选择性。此外，纯化的FLAG-Fmrp仅与一部分脑mRNA结合，包括髓鞘碱性蛋白信息及其自身信息的3'非翻译区。还纯化了缺少RGG框但保留两个KH结构域的重组异构体4，发现它仅与RNA弱相互作用。与之前的观点相反，携带严重脆性X综合征突变的FLAG纯化的I304N Fmrp表现出RNA结合。这些数据证明了Fmrp选择性结合RNA的固有特性，并表明FLAG-Fmrp是用于结构表征和鉴定同源RNA配体的合适试剂。
