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首页> 外文期刊>Molecular and Cellular Biology >Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.
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Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

机译：用单克隆抗体在体内表征异质核RNA-蛋白质复合物。

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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.
机译：将细胞暴露于足够强度的紫外线下会导致RNA交联到体内与其直接接触的蛋白质上。主要的[35S]蛋氨酸标记的蛋白质已与HeLa细胞中的聚腺苷酸异质核RNA交联，分子量分别为120,000（120K），68K，53K，43K，41K，38K和36K。纯化的聚腺苷酸RNA与完整细胞中通过紫外线交联获得的蛋白质形成的复合物可用于免疫小鼠，并产生针对其中一些蛋白质的单克隆抗体。用这些抗体表征了三种蛋白质（41K，43K和120K）的某些特性。 41K和43K多肽高度相关。它们被相同的抗体（2B12）识别，并具有相同的等电点（pI = 6.0 +/- 0.2），但具有不同的部分肽图。 41K和43K多肽是40S异质核糖核蛋白颗粒的一部分，并且似乎对应于先前描述的C蛋白（Beyer等，Cell II：127-138，1977）。另一种单克隆抗体（3G6）定义了一种新的120K主要异质核糖核蛋白。 41K，43K和120K多肽在体内与聚腺苷酸化和非聚腺苷酸化的核RNA都相关，并且所有三种蛋白质都被磷酸化了。单克隆抗体识别人和猴细胞中的相似蛋白，但不能识别其他几个脊椎动物中的相似蛋白。免疫荧光显微镜检查表明，这些蛋白质被隔离到细胞核，在细胞核中它们是细小颗粒非核仁结构的一部分。在用非离子去污剂原位提取的细胞中，所有41K和43K多肽都在盐浓度高达0.5 M NaCl时与细胞核结合，而120K多肽在此NaCl浓度下被完全提取。 41K和43K多肽的大部分（最多40％）保留在核基质中-一种对DNase I消化和2 M NaCl提取均具有抗性的结构，但41K和43K多肽被定量去除用RNase消化后加入0.5 M NaCl中。

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