van Eekelen C A, van Venrooij W J
J Cell Biol. 1981 Mar;88(3):554-63. doi: 10.1083/jcb.88.3.554.
In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.
在本研究中，从HeLa S3细胞中分离出DNA缺失的核蛋白基质。这些核基质由外周核纤层、残留核仁及内部纤维状结构组成。高分子量的不均一核RNA（hnRNA）定量地与这些结构相关联，并且只有通过影响基质的完整性才能完整释放。因此，可以得出结论，hnRNA是高度组织化核结构的一部分。通过用紫外线照射完整细胞或分离的核基质，与hnRNA紧密结合的蛋白质可被诱导与RNA交联。在体内进行交联可额外确保只有完整细胞中存在的hnRNA-蛋白质（hnRNP）复合物被共价连接。在能使非特异性RNA-蛋白质复合物完全解离的条件下分离并纯化此类hnRNP复合物。通过比较在核基质中发现的hnRNP与已发表的关于hnRNP颗粒组成的数据，发现所谓的hnRNP“包装”蛋白（分子量32,000 - 38,000）不能通过紫外线照射有效地与hnRNA交联。然而，它们存在于基质制剂中，与hnRNA结合，因为在对这些结构进行核糖核酸酶处理后它们从核基质中释放出来。另一方面，两种主要的hnRNP（分子量41,500和43,000）能有效地与hnRNA交联。这些蛋白质不会因核糖核酸酶处理而释放，这表明它们参与hnRNA与核基质的结合。
