Nuclear Matrix-like Filaments and Fibrogranular Complexes Form through the Rearrangement of Specific Nuclear Ribonucleoproteins

Abstract

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2) 3 B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to Ͼ10 m. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of Ͼ0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7-to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport. INTRODUCTIONWhen nuclei are extensively digested with DNase and RNase and extracted with high-salt solutions, an insoluble residue remains that retains the gross architecture of the nucleus (reviewed by Pederson, 1998Pederson, , 2000. The residue, termed the "nuclear matrix," represents a small percentage of the total nuclear protein mass, but it is very heterogeneous in protein composition (Hodge et al., 1977;Staufenbiel and Deppert, 1983;Kuzmina et al., 1984). The major proteins present in the residue are the nuclear lamins, proteins that constitute elements of the metaphase chromosome and interphase nuclear scaffold, nuclear envelope and pore complex proteins, and numerous proteins associated with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes (as discussed and referenced by Pederson, 1998).Among the numerous other proteins present in the residual material are those that have been hypothesized to form a fibrous network or fibrogranular complex (Berezney and Coffey, 1977;Kaufmann et al., 1981;Gallinaro et al., 1983). On the basis of various experimental observations it has been suggested that the matrix functions in interphase nuclei to bind DNA and organize chromatin (Basler et al., 1981;Brasch and Peters, 1985;Nickerson et al., 1997), that actively transcribing genes and nascent transcripts are associated with...

Recruitment of Heterogeneous Nuclear Ribonucleoprotein A1in Vivo to the LMP/TAP Region of the Major Histocompatibility Complex

“…for three independent PCR assays. component of the nuclear matrix, and of these, hnRNP-A1 is one of the most abundant (33). Expression of recombinant hnRNP-A1 and Western blot analysis of nuclear matrix extracts from MRC5 cells unambiguously showed that it is a nuclear matrix protein.…”

“…This network fills the nuclear interior and is connected to the nuclear lamina. Much debate has focused on whether the nuclear matrix is a structural and functional entity in itself, or whether it is induced during extraction procedures or cell fixation prior to electron microscopy examination (15,33). It has been suggested recently that, irrespective of the nature of the matrix in vivo, certain elements of the isolated matrix may reflect a significant "chemical footprint" of protein-protein and protein-nucleic acid interactions occurring during nuclear metabolism (15).…”

Polypurine-repeat-containing RNAs: a novel class of long non-coding RNA in mammalian cells

“…The components that provide structural integrity to the nuclear matrix remain enigmatic. Evidence indicates that the nuclear matrix comprises a highly ordered network of ribonucleoprotein (RNP) complexes, and in vitro studies have indicated that proteins like hnRNP A2 and B1 play crucial roles in the assembly of ordered filament-like nuclear structures (Nickerson, 2001;Tan et al, 2000). Because GRC-RNA interacts with hnRNP A2 and B1 and other nuclear matrix proteins, it is tempting to speculate a possible structural role for GRC-RNA in the formation of the core filament structure underlying nuclear matrix organization.…”

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