Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins.

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 >10 microm. 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 >0.1 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.

Trends in computational biology: a summary based on a RECOMB plenary lecture, 1999.

Computational biology, a term coined from analogy to the role of computing in the physical sciences, is now coming into its own as a major element of contemporary biological and biomedical research. Information science and computational science provide essential tools for next generation biological science efforts, from focusing the direction of experimental studies to providing knowledge and insight that can not otherwise be obtained. Going beyond the revolution in biology reflected in the successes of the genome project and driven by the power of molecular biology techniques, computational approaches will provide an underpinning for the integration of broad disciplines for development of a quantitative systems approach to understanding the mechanisms in the life of the cell.

Computational biology opportunity and challenges for the future.

Recent developments in high performance computers and computing methods have opened new avenues for tackling serious, important and challenging problems in biology and medicine. Only a few years back these problems were considered too complex and difficult, if not impossible to solve. An understanding of cross-disciplinary knowledge will be a prerequisite for applications of this enormous computing capability to enhance our understanding of governing principals in biology and medicine. We will show some specific research areas where computational biology can be applied effectively and then provide some ideas on future applications.

General organization of protein in HeLa 40S nuclear ribonucleoprotein particles.

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).

Polypeptide components of human small nuclear ribonucleoproteins.

Small nuclear RNA molecules (snRNAs) are associated with polypeptides in vivo, forming small nuclear ribonucleoprotein complexes (snRNPs). These snRNP complexes are targets for certain autoimmune antisera. Antisera of the type anti-Sm precipitate (and therefore define) a class including U1, U2, U4, U5, and U6 snRNAs, whereas antisera of the anti-RNP type precipitate only U1 snRNPs. We used these two types of autoimmune antisera (from patients with systemic lupus erythematosus) to study the polypeptide components in human cells. Sequential immunoprecipitation of the complexes from nuclear extracts with anti-RNP and anti-Sm antibodies, along with radioimmunoassay of protein transfers, identified four polypeptides of 14,000 (P14), 17,000 (P17), 26,000 (P26), and 27,000 (P27) daltons that are present on all members of this class, whereas a 68,000-dalton (P68) polypeptide is present only on U1 snRNPs. Based on the radioimmunoassay, three of these polypeptides, P17, P26, and P27, are also the antigens for anti-Sm antisera, whereas P68 is the antigen for anti-RNP antisera. Long-term phosphate labeling experiments show that the only detectably phosphorylated polypeptide is P68, which contains phosphoserine.

Small nuclear ribonucleoprotein complexes of Drosophila melanogaster.

We report here on the isolation and characterization of small nuclear ribonucleoproteins (snRNPs) and corresponding small nuclear RNA (snRNA) species from nuclei of Drosophila melanogaster. Velocity sedimentation in sucrose gradients was used to partially fractionate the RNPs; analysis of fractions so obtained suggests that, in general, one snRNP contains one snRNA. At least 11 species of snRNA are present in Drosophila nuclei; among them we identify a potential mammalian U1 homolog based on sequence homology. Autoimmune antiserum designated anti-Sm from patients with systemic lupus erythematosus recognizes nuclear antigens in Drosophila and precipitates seven species of snRNPs. The antigens in HeLa and Drosophila nuclei recognized by anti-Sm antibodies have been identified and compared. Anti-Sm antibodies at least bind to a 26,000-dalton polypeptide in HeLa extracts and to two polypeptides, one of 18,000 daltons and one of 26,000 daltons, in Drosophila extracts. This suggests that the 26,000-dalton polypeptide is an evolutionarily conserved antigenic component of Drosophila and HeLa snRNPs.

Isolation and characterization of nuclear ribonucleoprotein complexes from Drosophila melanogaster.

The isolation of total nuclear ribonucleoprotein particles from Drosophila melanogaster embryos, using a pH 8.0, 01 M NaCl extraction of purified nuclei, is described. When the extract is fractionated on isokinetic sucrose gradients, at least six major classes of nuclear ribonucleoprotein complexes, differing in RNA and protein content as well as sedimentation behavior, are observed. The two largest complexes are preribosomal complexes. The remaining four major classes of RNPs sediment at roughly 6S, 8S, 12S and 30S. A minor class at 17S is also observed. The 30S fraction is 200-250 A in width and appears to be analogous to the mammalian monoparticle. It is composed primarily of polypeptides at about 36 000 and 37 000 daltons, along with 1-2 kilobase RNA fragments. The 6S, 8S and 12S complexes contain a few discrete small nuclear RNAs from 80-600 bases in length, along with a small number of polypeptides, about 50 000, 52 000, 56 000 and 75 000 daltons. These novel complexes are of the order of a 100 A in width (60-120 A range).

The structure of the chromatin core particle in solution.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.

Low-angle neutron scattering from chromatin subunit particles.

Monomer chromatin particles containing 140 base pairs of DNA and eight histone molecules have been studied by neutron scattering. From measurements in various H2O/D2O mixtures, radii of gyration and the average scattering density of the particle were determined. The radius of gyration under conditions when scattering from the DNA dominates is 50A, and when scattering from the protein dominates, 30A. Consequently the core of the particle is largely occupied by the histones while the outer shell consists of DNA together with some of the histone.

Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-1-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core.