Cunningham K S, Dodson R E, Nagel M A, Shapiro D J, Schoenberg D R
Department of Molecular and Cellular Biochemistry, The Comprehensive Cancer Center, and Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA.
Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12498-502. doi: 10.1073/pnas.220425497.
In Xenopus, estrogen induces the stabilization of vitellogenin mRNA and the destabilization of albumin mRNA. These processes correlate with increased polysomal activity of a sequence-selective mRNA endonuclease, PMR-1, and a hnRNP K homology-domain RNA-binding protein, vigilin. Vigilin binds to a region of the vitellogenin mRNA 3'-untranslated region (3'-UTR) implicated in estrogen-mediated stabilization. The vigilin-binding site in the vitellogenin B1 mRNA 3'-UTR contains two consensus PMR-1 cleavage sites. The availability of purified PMR-1 and recombinant vigilin made it possible to test the hypothesis that RNA-binding proteins interact with cis-acting elements to stabilize target mRNAs by blocking cleavage by site-specific mRNA endonucleases. Vigilin binds to the vitellogenin mRNA 3'-UTR site with at least 30-fold higher affinity than it exhibits for the albumin mRNA segment containing the mapped PMR-1 cleavage sites. This differential binding affinity correlates with differential in vitro susceptibility of the protein-RNA complexes to cleavage by PMR-1. Whereas recombinant vigilin has no detectable protective effect on PMR-1 cleavage of albumin mRNA, it retards in vitro cleavage of the vitellogenin mRNA 3'-UTR by purified PMR-1. The PMR-1 sites in the vitellogenin mRNA 3'-UTR are functional because they are readily cleaved in vitro by purified PMR-1. These results provide direct evidence for differential susceptibility to endonuclease-mediated mRNA decay resulting from the differential affinity of a RNA-binding protein for cis-acting stability determinants.
在非洲爪蟾中，雌激素可诱导卵黄蛋白原mRNA的稳定以及白蛋白mRNA的降解。这些过程与一种序列选择性mRNA核酸内切酶PMR-1和一种异质性核糖核蛋白K同源结构域RNA结合蛋白vigilin的多核糖体活性增加相关。Vigilin与卵黄蛋白原mRNA 3'-非翻译区（3'-UTR）中与雌激素介导的稳定有关的区域结合。卵黄蛋白原B1 mRNA 3'-UTR中的vigilin结合位点包含两个共有PMR-1切割位点。纯化的PMR-1和重组vigilin的可得性使得能够检验这样一种假说，即RNA结合蛋白通过阻断位点特异性mRNA核酸内切酶的切割作用与顺式作用元件相互作用以稳定靶mRNA。Vigilin与卵黄蛋白原mRNA 3'-UTR位点的结合亲和力比其对含有已定位PMR-1切割位点的白蛋白mRNA片段的亲和力至少高30倍。这种差异结合亲和力与蛋白质-RNA复合物在体外对PMR-1切割的不同敏感性相关。虽然重组vigilin对白蛋白mRNA的PMR-1切割没有可检测到的保护作用，但它能延缓纯化的PMR-1对卵黄蛋白原mRNA 3'-UTR的体外切割。卵黄蛋白原mRNA 3'-UTR中的PMR-1位点是有功能的，因为它们在体外很容易被纯化的PMR-1切割。这些结果为由于RNA结合蛋白对顺式作用稳定决定因素的差异亲和力而导致的对核酸内切酶介导的mRNA降解的不同敏感性提供了直接证据。
