ISOLATION OF THE HETEROGENEOUS NUCLEAR-RNA RIBONUCLEOPROTEIN COMPLEX (HNRNP) - A UNIQUE SUPRAMOLECULAR ASSEMBLY
ISOLATION OF THE HETEROGENEOUS NUCLEAR-RNA RIBONUCLEOPROTEIN COMPLEX (HNRNP) - A UNIQUE SUPRAMOLECULAR ASSEMBLY
复制标题
DOI：
10.1073/pnas.81.23.7471
复制
发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
DREYFUSS, G
中科院分区：
文献类型：
--
作者：
CHOI, YD;DREYFUSS, G
据信，异质核RNA（hnRNA）的包装、hnRNA在核中的命运以及hnRNA向mRNA的转化发生在hnRNA转录物与特定蛋白质结合以形成称为hnRNP复合物的核糖核蛋白复合物时。hnRNP的蛋白质成分的身份和组织一直是一个相当大的争议。从脊椎动物细胞核的hnRNP复合物的分离报道，采用免疫沉淀与单克隆抗体对主要蛋白质，在HeLa细胞中与hnRNA接触。用针对hnRNP C蛋白的2种不同单克隆抗体（41和43 kDa [千道尔顿]）从HeLa核质中快速免疫沉淀分离出类似的复合物，该复合物含有高达约100 kDa的蛋白质和hnRNA。10种酶。分离的复合物的主要稳态[35 S]甲硫氨酸标记蛋白为34 kDa、36 kDa（A1和A2）、37 kDa、38 kDa（B1和B2）、41 kDa、43 kDa（C1和C2），以及68 kDa和120 kDa的双联体。还观察到从45 kDa到非常高分子量的其他蛋白质。通过NaDodSO 4/聚丙烯酰胺凝胶电泳，复合物的主要蛋白质与真正的hnRNP蛋白质相同，这些蛋白质在体内通过UV光与hnRNA交联。免疫沉淀与不同的，noncrossreacting单克隆抗体的120-kDa的蛋白质分离出一个明显相同的复合物的蛋白质，存在于一个类似的相对化学计量。在啮齿动物[仓鼠]和禽类[鸡]细胞中发现了类似的hnRNP复合物。核酸酶双链反应表明RNA在维持结构的完整性中起作用，并且完整的RNA 125个核苷酸足以将蛋白质复合物结合在一起。hnRNA和所有的蛋白质，通过抗体对不同的真正的hnRNP蛋白质和从不同的物种的免疫共沉淀强烈表明，hnRNP复合物是一个单一的结构一致，定义和保守的组件。
The packaging of heterogeneous nuclear RNA (hnRNA), the fate of hnRNA in the nucleus, and the conversion of hnRNA to mRNA are believed to occur as the hnRNA transcript is associated with specific proteins to form a ribonucleoprotein complex termed the hnRNP complex. The identity and organization of the protein constituents of the hnRNP have been a matter of considerable controversy. The isolation of the hnRNP complex from vertebrate cell nuclei is reported, employing immunoprecipitation with monoclonal antibodies against the major proteins that are in contact with hnRNA in HeLa cells. Rapid immunoprecipitation from HeLa nucleoplasm with 2 different monoclonal antibodies to the hnRNP C proteins (41 and 43 kDa [kilodaltons] isolates similar complex that contains proteins and hnRNA of up to .apprxeq. 10 kilobases. The major steady-state [35S]methionine-labeled proteins of the isolated complex are of 34 kDa, 36 kDa (A1 and A2), 37 kDa, 38 kDa (B1 and B2), 41 kDa, 43 kDa (C1 and C2), and doublets at 68 kDa and at 120 kDa. Additional proteins from 45 kDa to very high molecular mass are also seen. The major proteins of the complex appear identical by NaDodSO4/polyacrylamide gel electrophoresis to genuine hnRNP proteins, those which become crosslinked by UV light to the hnRNA in vivo. Immunoprecipitation with a different, noncrossreacting monoclonal antibody to the 120-kDa protein isolates an apparently identical complex of proteins that are present at a similar relative stoichiometry. Similar hnRNP complexes are found in rodent [hamster] and avian [chicken] cells. Nuclease digestions indicate that RNA plays a role in maintaining the integrity of the structure and that intact RNA of .apprxeq. 125 nucleotides is sufficient to hold the complex of proteins together. The coimmunoprecipitation of the hnRNA and of all of the proteins through antibodies against different genuine hnRNP proteins and from divergent species strongly suggests that the hnRNP complex is a unitary structure of consistent, defined and conserved components.