Wallace M. LeStourgeon

Identification and characterization of the packaging proteins of core 40S hnRNP particles

Abstract The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C 1 and C 2 of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A 1 and A 2 of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N G ,N G -dimethylarginine. The major particle proteins (A 1 and A 2 ) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A 1 , A 2 and B 1 ) is consistent with a conserved particle structure and function in eucaryotes.

The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.

Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles.

A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote Physarum polycephalum, purified, and found to contain a substantial amount of the unusual amino acid NG, NG-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in Physarum polycephalum and the possible significance of the NG, NG-dimethylarginine are discussed.

Chapter 26 The Rapid Isolation, High-Resolution Electrophoretic Characterzation, and Purification of Nuclear Proteins

Publisher Summary This chapter describes the methods for the rapid isolation, high-resolution electrophoretic characterization, and purification of nuclear proteins. The chapter discusses a procedure for solubilizing “total” protein either from whole nuclei, from subnuclear preparations, or from various chromatographic or density-gradient fractions. The procedure yields nondegraded protein free of nucleic acid and polysaccharide contaminants, and following a single dialysis it is ready to load on high-resolution polyacrylamide gels. The electrophoretic procedures are also described. The procedures allow the direct visualization and quantitation of hundreds of polypeptides in submicrogram amounts. Selected procedures for obtaining purified proteins so that more complete characterizations of individual proteins can be obtained are outlined. The methods described for purifying specific polypeptides are reliable but yield denatured protein. The procedures outlined in the chapter can be instrumental in monitoring the progress of attempts to characterize nuclear proteins through more classical biochemical techniques. The methods for the protein purification through preparative sodium dodecyl sulfate (SDS)-gel electrophoresis and isoelectric focusing are also discussed.

The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2

The C proteins (C1 and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S.F. Barnett, W.M. LeStourgeon, and D.L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume of 0.70 cm3/g based on the amino acid composition, the molecular weight of the complex was calculated to be 135,500. This corresponds well to 129,056, the sequence-determined molecular weight of a (C1)3C2 tetramer. Reversible chemical cross-linking with dithiobis(succinimidyl propionate) and analysis of cross-linked and cleaved complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the C proteins exist as tetramers, most or all of which are composed of (C1)3C2. The tetramer is stable in a wide range of NaCl concentrations (0.09 to 2.0 M) and is not dissociated by 0.5% sodium deoxycholate. This stability is not the result of disulfide bonds or interactions with divalent cations. The hydrodynamic properties of highly purified C-protein tetramers are the same for C-protein complexes released from intact particles with RNase or high salt. These findings support previous studies indicating that the core particle protein stoichiometry of 40S heterogeneous nuclear ribonucleoproteins is N(3A1-3A2-1B1-1B2-3C1-1C2), where N = 3 to 4, and demonstrate that the C-protein tetramer is a fundamental structural element in these RNA-packaging complexes. The presence of at least three tetramers per 40S monoparticle, together with the highly anisotropic nature of the tetramer, suggesting that one-third of the 700-nucleotide pre-mRNA moiety packaged in monoparticles is associated through a sequence-independent mechanism with the C protein.

Identification of proliferation-sensitive human proteins amongst components of the 40 S hnRNP particles: Identity of hnRNP core proteins in the HeLa protein catalogue

The core proteins of HeLa 40 S hnRNP monoparticles have been identified in the HeLa protein catalogue. Human proteins previously indemnified as proliferation‐sensitive [NEPHGE 21 and 17; Bravo, R. and Celis, J.E. (1982) Clin. Chem. 28, 766], as well as two proteins characterized in this study (NEPHGE 16 W and 16 Wl), are shown to be components of these particles. These basic nuclear polypeptides correspond to core proteins al, Bla, B2 and C4, respectively. The significance of these results in terms of composition and function of hnRNP particles is discussed.

The Heterogeneous Nuclear Ribonucleoprotein C Protein Tetramer Binds U1, U2, and U6 snRNAs through Its High Affinity RNA Binding Domain (the bZIP-like Motif)

Based on UV cross-linking experiments, it has been reported that the C protein tetramer of 40 S heterogeneous nuclear ribonucleoprotein complexes specifically interacts with stem-loop I of U2 small nuclear RNA (snRNA) (Temsamani, J., and Pederson, T. (1996)J. Biol. Chem. 271, 24922–24926), that C protein disrupts U4:U6 snRNA complexes (Forne, T., Rossi, F., Labourier, E., Antoine, E., Cathala, G., Brunel, C., and Tazi, J. (1995) J. Biol. Chem. 270, 16476–16481), that U6 snRNA may modulate C protein phosphorylation (Mayrand, S. H., Fung, P. A., and Pederson, T. (1996) Mol. Cell. Biol. 16, 1241–1246), and that hyperphosphorylated C protein lacks pre-mRNA binding activity. These findings suggest that snRNA-C protein interactions may function to recruit snRNA to, or displace C protein from, splice junctions. In this study, both equilibrium and non-equilibrium RNA binding assays reveal that purified native C protein binds U1, U2, and U6 snRNA with significant affinity (∼7.5–50 nm) although nonspecifically. Competition binding assays reveal that U2 snRNA (the highest affinity snRNA substrate) is ineffective in C protein displacement from branch-point/splice junctions or as a competitor of C protein’s self-cooperative RNA binding mode. Additionally, C protein binds snRNA through its high affinity bZLM and mutations in the RNA recognition motif at suggested RNA binding sites primarily affect protein oligomerization.

Isolation and in vitro assembly of nuclear ribonucleoprotein particles and purification of core particle proteins

Publisher Summary This chapter describes the isolation and in vitro assembly of nuclear ribonucleoprotein particles and purification of core particle proteins. In order to obtain hnRNP complexes which are not contaminated with ribosomes or other cytoplasmic components it is very important to obtain rapidly nuclei free of cytoplasmic material. Native C-protein tetramers can be purified from isolated 40 S hnRNP particles through a two-step procedure using gel filtration in 350 mM NaCI and anion-exchange chromatography. Many questions regarding the composition, structure, and function of hnRNP complexes have been difficult to answer experimentally because the RNA component of isolated monoparticles is as heterogeneous as the entire complement of nuclear pre-mRNA and because the core particle proteins do not protect the packaged length of RNA from endogenous nuclease action. Although isolated 40 S hnRNP particles will dissociate in 0.7 M NaC1 and spontaneously reassemble on back-dialysis into low salt, this procedure is complicated by the necessity of removing the endogenous RNA fragments under high-salt conditions to prevent particle reassembly.

Rapid purification of native C protein from nuclear ribonucleoprotein particels

A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.

Chapter 16 Identification of Contractile Proteins in Nuclear Protein Fractions

Publisher Summary This chapter reviews the biochemical and cytological evidence for actin and myosin in nuclei and nucleoli of Physarum polycephalum obtained by Jockusch and colleagues. The chapter describes the identification of contractile proteins in nuclear protein fractions and emphasizes on methods for the rapid identification of actin and myosin in nuclear protein fractions. . The actin–myosin interaction in the force-producing mechanism for chromosome movement is discussed. The procedures for confirmation of actin in nuclear protein fractions are the most direct, unequivocal, and least time-consuming. For myosin identification the most important aspects are the tail-to-tail associations of myosin, the highly specific interaction of actin with myosin, and the stimulation of adenosine triphosphate (ATP) hydrolysis that follows this interaction. In addition to these two major components of contractile systems, the regulatory proteins tropomyosin and troponin are likely to be present. There is evidence for tropomyosin in rat liver chromatin. However, it is illogical to attempt identification of these proteins before actin confirmation. Methods for purifying these components are more complex than the methods for actin and myosin, as are the procedures for confirmation and characterization.