Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Major internal nuclear matrix proteins are common to different human cell types.

The nuclear matrix may be involved in the structural and functional organization of the cell nucleus. However, we still do not understand the molecular basis of the intranuclear fibrogranular network that is part of the nuclear matrix. We recently described a method to identify internal nuclear matrix proteins [Mattern et al. (1996): J Cell Biochem 62:275-289], which was done by comparing two nuclear matrix preparations: one with and one without the internal structure by using quantitative two-dimensional gel electrophoresis. In the present study, we use the same approach to compare the nuclear matrix proteins of four different human cell types to investigate whether they have a similar internal nuclear matrix protein composition. Major nuclear matrix proteins present in all these cell types likely represent the base of the internal nuclear matrix. We demonstrate that the 25 most abundant internal nuclear matrix proteins are common to all four cell types. Together, these common proteins represent more than 75% of the total internal nuclear matrix protein mass in each cell type. This set of proteins includes B23 and most hnRNP proteins. The quantity of most of these proteins is very similar in the four cell types. The fact that the internal nuclear matrix consists mainly of hnRNP proteins, which may be involved in transcription, transport, and processing of hnRNA, supports the idea that the internal nuclear matrix is the result of these processes.

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells.

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa 53 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.

Nuclear domains involved in RNA synthesis, RNA processing, and replication.

Two main principles of nuclear organization have been outlined on the basis of contributions by many research groups in recent years. The first principle is that interphase chromosomes occupy discrete territories in the nucleus, with no intermingling of the DNA from different chromosomes. Within a chromosome territory the DNA is organized in chromatin fibers at several levels of folding, that meander through the territory. Transcription and replication take place at the surface of these higher order chromatin fibers, probably on locally unfolded DNA templates. The second principle is that different types of nuclear domains are associated with several specific gene loci. This holds for clusters of interchromatin granules, coiled bodies, RNA 3'-cleavage factor-containing nuclear bodies (cleavage bodies) and probably PML-containing nuclear bodies. These domains may play an important role in the spatial arrangement of genes in the interphase nucleus. Despite these new insights, our knowledge of the function of many nuclear compartments and the molecular interactions responsible for the dynamic organization of a compartmentalized nucleus is still in its infancy.