Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.
Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.
复制标题
使用单克隆抗体表征体内异质核 RNA-蛋白质复合物。
DOI：
10.1128/mcb.4.6.1104-1114.1984
复制
发表时间：
1984
影响因子：
5.3
通讯作者：
Adam,SA
中科院分区：
文献类型：
--
作者：
Dreyfuss,G;Choi,YD;Adam,SA
将细胞暴露在足够强度的紫外线下，会使RNA与体内与其直接接触的蛋白质发生交联。HeLa细胞中与聚腺苷化异质核RNA交联的主要[35S]蛋氨酸标记蛋白分子量为120,000 （120K）、68K、53K、43K、41K、38K和36K。在完整细胞中通过UV交联获得纯化的多腺苷化RNA与蛋白质的复合物，用于免疫小鼠并产生针对这些蛋白质的单克隆抗体。其中三种蛋白（41K、43K和120K）的一些特性可以用这些抗体表征。41K和43K多肽高度相关。它们被相同的抗体（2B12）识别，具有相同的等电点（pl = 6.0±0.2），但部分肽图不同。41K和43K多肽是40S异质核核糖核蛋白颗粒的一部分，似乎与先前描述的C蛋白相对应（Beyer et al., cell11:127 - 138,1977）。一个不同的单克隆抗体（3G6）定义了一个新的主要的异质核糖核蛋白120K。41K， 43K和120K多肽在体内与聚腺苷化和非聚腺苷化的核RNA相关，并且这三种蛋白都被磷酸化。这种单克隆抗体在人类和猴子细胞中识别出类似的蛋白质，但在其他几种脊椎动物细胞中却没有。免疫荧光显微镜显示，这些蛋白质被分离到细胞核，在那里它们是一个细颗粒非核仁结构的一部分。在非离子型洗剂原位提取的细胞中，当盐浓度达到0.5 M NaCl时，41K和43K的多肽全部与细胞核结合，而120K的多肽在此NaCl浓度下完全被提取。大部分41K和43K多肽（高达40%）保留在核基质中，这种结构可以抵抗DNase I的消化和2 M NaCl的萃取，但在RNase的消化后，41K和43K多肽在0.5 M NaCl的条件下被定量去除。
Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 ± 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell11:127–138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix—a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.