Montaville P, Dai Y, Cheung C Y, Giller K, Becker S, Michalak M, Webb S E, Miller A L, Krebs J
Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Biochim Biophys Acta. 2006 Nov;1763(11):1335-43. doi: 10.1016/j.bbamcr.2006.09.003. Epub 2006 Sep 14.
By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.
通过以钙结合蛋白ALG-2作为诱饵进行酵母双杂交筛选，确定了ALG-2的一个新靶点——RNA结合蛋白RBM22。为了在体内证实这些相互作用，我们制备了荧光构建体，用单体红色荧光蛋白标记ALG-2，用增强型绿色荧光蛋白标记RBM22。对单独转染编码荧光融合蛋白的ALG-2或RBM22表达构建体的NIH 3T3细胞进行共聚焦显微镜观察发现，大多数ALG-2定位于细胞质中，而RBM22定位于细胞核中。当细胞同时转染编码两种融合蛋白的表达载体时，发现ALG-2存在于细胞核中，这表明可以在细胞质和细胞核之间穿梭的RBM22可能在ALG-2的核转运中发挥作用。以斑马鱼为模型，在1细胞期将ALG-2和RBM22的mRNA同源物显微注射到斑马鱼胚胎的胚盘-卵黄边缘，然后在斑马鱼发育过程中监测融合蛋白。据此，我们观察到单独的ALG-2在细胞内均匀分布，而在有RBM22存在的情况下，这两种蛋白在细胞核内共定位。超过95%的两种蛋白在细胞核的同一区域共定位，这表明Ca(2+)信号蛋白ALG-2和RNA结合蛋白RBM22之间存在功能相互作用。
