Dreyfuss G, Choi Y D, Adam S A
Mol Cell Biol. 1984 Jun;4(6):1104-14. doi: 10.1128/mcb.4.6.1104-1114.1984.
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., Cell II: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.
将细胞暴露于足够强度的紫外线下会导致RNA与体内与其直接接触的蛋白质发生交联。在HeLa细胞中与多聚腺苷酸化的不均一核RNA发生交联的主要[35S]甲硫氨酸标记的蛋白质，其分子量分别为120,000（120K）、68K、53K、43K、41K、38K和36K。通过完整细胞中的紫外线交联获得的多聚腺苷酸化RNA与蛋白质的纯化复合物被用于免疫小鼠，并产生针对其中几种蛋白质的单克隆抗体。利用这些抗体对其中三种蛋白质，即41K、43K和120K的一些特性进行了表征。41K和43K多肽高度相关。它们被同一种抗体（2B12）识别，具有相同的等电点（pl = 6.0 +/- 0.2），但部分肽图谱不同。41K和43K多肽是40S不均一核核糖核蛋白颗粒的一部分，似乎对应于先前描述的C蛋白（Beyer等人，《细胞》11:127 - 138，1977）。另一种单克隆抗体（3G6）确定了一种新的主要的120K不均一核糖核蛋白。41K、43K和120K多肽在体内与多聚腺苷酸化和非多聚腺苷酸化的核RNA都有关联，并且这三种蛋白质都被磷酸化。这些单克隆抗体在人和猴细胞中识别相似的蛋白质，但在其他几种脊椎动物细胞中则不能。免疫荧光显微镜显示这些蛋白质定位于细胞核，它们是一种精细的非核仁颗粒结构的一部分。在用非离子去污剂原位提取的细胞中，在盐浓度高达0.5M NaCl时，所有的41K和43K多肽都与细胞核相关联，而120K多肽在此NaCl浓度下被完全提取。相当一部分的41K和43K多肽（高达40%）与核基质结合——核基质是一种对DNase I消化和2M NaCl提取具有抗性的结构，但在用RNase消化后，41K和43K多肽在0.5M NaCl时被定量去除。
