Ultrastructural Organization and Biochemical Characterization of Chromatin · RNA · Protein Complexes Isolated from Mammalian Cell Nuclei

Abstract

Extranucleolar elements were isolated from mammalian cell nuclei and characterized by correlative biochemical-ultrastructural studies. These complex structures were shown to retain a supra-particle arrangement closely resembling the organizaton in situ of definite nuclear areas. In particular the morphological RNA . protein species, namely perichromatin fibrils and granules, were detected in association with characteristic regions of chromatin. By autoradiographic experiments, we were able to show that perichromatin fibrils represent the morphological state of newly formed heterogeneous nuclear RNA (hnRNA).Chromatin . RNA . protein complexes were further fractionated by means of a treatment dissociating the chromatin component. A minor part of DNA, possibly involved in the biosynthesis and the processing of hnRNA, remains linked to the resulting RNA . protein network.Most of the mechanisms involved in the control of RNA maturation in eukaryotic cells still remain to be elucidated. It has now been clearly established that a class of heterogeneous nuclear RNA (hnRNA) of high molecular weight, rapidly labeled and with a DNA-like composition [l-'51, presents The present study was undertaken in order to establish tentative correlation between biochemical and ultrastructural features of nuclear RNA protein particles and their spatial organization in situ.Abbreviation. hnRNA, heterogeneous nuclear RNA. ~9 1 .We developed an isolation procedure of subnuclear fractions where the ultrastructural organization of definite RNA . protein . chromatin areas appears well preserved. This method could contribute to the characterization of actively transcribed zones in chromatin as well as to a better definition of the events involved in hnRNA metabolism. We here report direct evidence that perichromatin fibrils do indeed represent the morphological state of newly synthesized hnRNA, as previously assumed by autoradiographic studies in situ [18]. MATERIALS AND METHODS Cell Culture and LabelingZadjela Ascitic Hepatoma Cells. Female Wistar rats were inoculated intraperitoneally with approximately 2 x lo7 cells. After 3 days, the cells were collected, washed repeatedly in a phosphate-buffered saline medium (NaC10.12 M, KCl4 mM, Na2HP04 7 mM, KH2P0, 1 mM, NaHCO, 16 mM) and resuspended in Fischer [19] medium (Eurobio), supplemented with 5 % calf serum, 100 U/ml penicillin and 50 pg/ml streptomycin. The cell suspension was then incubated at 37 "C in glass flasks at concentrations varying from 5 x 105/ml to 106/ml according to the experiments planned. Cells were labeled after a 5-h incubation in synthetic medium by addition of radioactive precursors.

U1 and U2 small nuclear RNAs are present in nuclear speckles.

“…We have localized U1 and U2 snRNAs to the same speckled nuclear regions (perichromatin fibrils and interchromatin granules) where snRNP antigens (3-9) and the essential splicing factor SC-35 (14,16) have been localized. These speckles interconnect to form a three-dimensional network in the nucleus (6,16 (17,18), which are a component of the snRNP speckled network (16). These studies suggest that the speckled regions enriched in splicing components play a role in pre-mRNA splicing.…”

“…This speckled distribution pattern observed by fluorescence microscopy represents nuclear substructures termed interchromatin granules and perichromatin fibrils (16). Perichromatin fibrils are thought to represent RNA transcripts (17,18), while interchromatin granules may represent RNP storage (19,20) and/or assembly sites (16). To address the question of whether the U1 and U2 snRNAs are also part of the same staining regions, we compared the localization of U1 and U2 snRNAs with the localization of snRNP antigens (data not shown) and SC-35 (Fig.…”

Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol

“…Most of the sites of non-nucleolar transcription are located at these sites, especially at the periphery of compact or semi-condensed chromatin (Fakan and Bernhard, 1971;Bachellerie et al, 1975;Cmarko et al, 1999;Puvion and Puvion-Dutilleul, 1996;Fakan, 2004). The association of R-RAF with structures related to the process of transcription supports the proposition that the naER-E-RAF heterodimer binds to RNA-polymerase II at the site of transcription initiation (Govind and Thampan, 2001).…”

“…1975;Bachellerie et al, 1975;Fakan and Bernhard, 1971;Fakan et al, 1976;Fakan et al, 1984;Puvion et al, 1984;Spector, 1984;Fakan et al, 1986;Cmarko et al, 1999;Fakan, 2004). Our results demonstrate that the complex of snRNPs containing and nER II binding snRNPs p32, p55 and p60 is located at perichromatin fibrils and thus that these proteins may be related to active spliceosomes.…”

Nucleolar Chromatin in Chinese Hamster Ovary Cells

“…Mg2+ concentration was then lowered to 3 mM, by addition of an equal volume of buffer B (identical to A except for 0.5 mM MgC12) and an additional sonication step was carried out in order to achieve the dispersion of most extra-nucleolar chromatin aggregates while the nucleolar structures were preserved. Fractionation of the dispersed nuclear components and isolation of purified nucleoli and non-nucleolar chromatin were carried out by a series of differential centrifugations, according to previously described procedures [8,19]. Briefly, nucleoli were recovered from the sonicated suspension by a 5-min centrifugation at 500 x g resuspended in buffer C (10 mM Tris-HCl pH 7.4, 20 mM KCl, 2 mM MgC12, 20 pg/ml polyvinyl sulfate) and collected by a 5-min centrifugation at 1500 x g.…”