Vedeler A, Hollås H
Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
Biochem J. 2000 Jun 15;348 Pt 3(Pt 3):565-72.
Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.
蛋白质与mRNA的相互作用会影响mRNA在细胞质中的运输、锚定、稳定性和可翻译性。在纯化三个多核糖体亚群的过程中，观察到一种36 kDa的蛋白质（被鉴定为膜联蛋白II）仅与一个特定的多核糖体群体相关联，即与细胞骨架相关的多核糖体。这种关联似乎依赖于钙，因为它对乙二醇双四乙酸（EGTA）敏感，并且可以在体外重建。紫外线照射导致膜联蛋白II与多核糖体发生部分的、对EGTA有抗性的交联。在蛋白质印迹法中，将（32）P标记的总RNA与从细胞骨架结合的多核糖体中分离出的蛋白质结合，产生了一条与膜联蛋白II迁移率相同的放射性条带，最重要的是，固定在硝酸纤维素上的纯化天然膜联蛋白II能特异性结合mRNA。与从游离或膜结合的多核糖体中分离出的mRNA群体相比，从细胞骨架结合的多核糖体中分离出的mRNA群体与膜联蛋白II的结合亲和力最高。有趣的是，从猪小肠微绒毛中分离出的膜联蛋白II复合物比从转化的克雷布斯II腹水细胞中获得的复合物是更好的mRNA结合底物。当细胞骨架相关的多核糖体被分成60 S和40 S核糖体亚基以及一个含有mRNA复合物的峰时，膜联蛋白II与mRNA一起分级分离。最后，利用在聚（A）（+）偶联磁珠上对mRNA进行亲和纯化，仅在细胞骨架部分（非多核糖体mRNA）中存在的信使核糖核蛋白（mRNP）中检测到膜联蛋白II。这些来自体外实验和细胞分级分离的结果表明，膜联蛋白II直接与含有特定mRNA群体的mRNP复合物的RNA部分结合。
