The core proteins A2 and B1 exist as (A2)3B1 tetramers in 40S nuclear ribonucleoprotein particles.

The six "core" proteins of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) package 700-nucleotide lengths of pre-mRNA into a repeating array of regular particles. We have previously shown that the C proteins exist as anisotropic tetramers of (C1)3C2 in 40S hnRNP particles and that each particle probably contains three such tetramers. We report here that proteins A2 and B1 also exist in monoparticles as (A2)3B1 tetramers and that each monoparticle contains at least three such tetramers. Proteins A2 and B1 dissociate from isolated monoparticles as a stable tetramer upon nuclease digestion. In low-salt gradients, the tetramers sediment at 6.8S, which is consistent with a mass of 145 kDa. In 200 mM salt, the concentration which dissociates these proteins from RNA, only 4.2S dimers exist in solution. Tetramers of (A2)3B1 possess the ability to package multiples of 700 nucleotides of RNA in vitro into an array of regular, 22.5-nm 43S particles. Unlike the in vitro assembly of intact 40S hnRNP, the (A2)3B1 tetramers assemble by means of a highly cooperative process. These findings indicate that the (A2)3B1 tetramers play a major role in hnRNP assembly and they further support the contention that 40S monoparticles are regular structures composed of three copies of three different tetramers, i.e., 3[(A1)3B2, (A2)3B1, (C1)3C2].     

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 identified 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 W1), are shown to be components of these particles. These basic nuclear polypeptides correspond to core proteins A1, B1a, B2 and C4, respectively. The significance of these results in terms of composition and function of hnRNP particles is discussed.     

Variation in the glycosylation of the murine sex-limited protein: comparison with the fourth component of murine complement

The murine sex-limited protein (Slp) is a hemolytically nonfunctional homologue of the fourth component of complement (C4). Two congenic mouse strains, B10.BUA1 (H-2w16) and B10.KPB128 (H-2w19), which have been previously shown to share a variant form of C4 (Karp et al., J. Biol. Chem., 257: 7330-7335), were examined and found to also produce a variant form of Slp. Slp molecules isolated from the plasma or peritoneal macrophage cultures from these strains have an alpha-chain approximately 2,000 daltons smaller than the alpha-chain of Slp from H-2d or H-2w7 mice as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis. Expression of this Slp was constitutive, i.e., not regulated by androgen, and is cis-dominant in F1 hybrid mice. Autolysis of the different relative molecular mass (Mr) alpha-chains at the internal thiolester produced similar Mr amino-terminal fragments and different Mr carboxy-terminal fragments. Deglycosylation of the alpha-chains with trifluoromethanesulfonic acid eliminated most, if not all, the Mr difference. The Mr difference was also manifested by the intracellular precursors of Slp and could be eliminated by endoglycosidase H (endo H) treatment. The number of oligosaccharides on the Slp alpha-chain was deduced by limited endo H treatment of Slp synthesized in the presence of swainsonine, a plant alkaloid that prevents maturation of complex-type oligosaccharides. This method is a simple way to enumerate the complex-type, N-linked oligosaccharides on glycoproteins. The genetic variation in the glycosylation of Slp was compared with the known variation in glycosylation of C4, and a scheme depicting some of the structural differences among these molecules was developed.     

Rapid purification of native C protein from nuclear ribonucleoprotein particles

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.     

Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins

Protein A1 (Mr approximately 32,000), a major glycine-rich protein of heterogeneous nuclear ribonucleoproteins (hnRNP), was purified to near homogeneity under nondenaturing conditions from HeLa cells. Limited proteolysis of the native protein yields a trypsin-resistant N-terminal nucleic acid-binding domain about 195 amino acids long which has a primary structure nearly identical to that of the 195-amino acid-long single-stranded DNA (ssDNA)-binding protein UP1 (Mr 22,162) from calf thymus (Williams, K.R., Stone, K. L., LoPresti, M.B., Merrill, B. M., and Planck, S.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670). 45 of the 61 glycine residues of A1 are present in the trypsin-sensitive C-terminal domain of the protein which contains no sequences homologous to UP1. Protein A2, another major glycine-rich core hnRNP protein from HeLa, has a domain structure analogous to A1 and appears to be related to ssDNA-binding proteins UP1-B from calf liver and HDP-1 from mouse myeloma in a way similar to the A1/UP1 relationship. In contrast to ssDNA-binding proteins, A1 binds preferentially to RNA over ssDNA and exhibits no helix-destabilizing activity.     

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.     

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.     

Single stranded DNA binding proteins derive from hnRNP proteins by proteolysis in mammalian cells.

As we have previously demonstrated, mammalian single stranded DNA binding proteins (ssDBP) and heterogeneous nuclear RNA binding proteins (hnRNP proteins) are antigenically and structurally related. In this paper we show that ssDBP are specific proteolytic products of hnRNP core proteins. Proteolysis can be observed in crude extract, both total and nuclear and is not inhibited by the most commonly used protease inhibitors. Such phenomenon can be observed in HeLa cells, human fibroblasts and calf thymus extracts. A trypsin-like protease that cleaves purified hnRNP proteins to give ssDBP of Mr = 24-28 Kd can be purified from HeLa cells. A precursor-product relationship can be established between hnRNP core proteins type A and an ssDBP of 24 Kd (UP1).     

A detergent-activated tyrosinase from Xenopus laevis. I. Purification and partial characterization

A tyrosinase has been purified from the skin of the frog Xenopus laevis. Dihydroxyphenylalanine oxidase and tyrosine hydroxylase activities co-purify throughout the procedure. The enzyme is isolated in an inactive form, but both enzymatic activities are activated by a variety of anionic detergents. Of these, sodium dodecyl sulfate (NaDodSO4) is the most effective. The enzyme activation occurs at NaDodSO4 concentrations well below the critical micelle concentration and it remains active at concentrations as high as 30 mM (1%). Neither activity is stimulated by cationic or nonionic detergents, or a variety of other agents, including trypsin. The purified tyrosinase is a glycoprotein having a polypeptide Mr = 175,000 by NaDodSO4-polyacrylamide gel electrophoresis. This monomeric species is enzymatically active in the presence of NaDodSO4. Detergent-activated tyrosinase has a KM for dihydroxyphenylalanine of 6 X 10(-4) M and a KM for tyrosine of 4 X 10(-4) M. Both activities are inhibited by copper chelators but not by an iron chelator. Further characterization of the detergent activation of this enzyme is presented in a companion paper (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12542-12546).     

RNA binding specificity of hnRNP proteins: a subset bind to the 3'' end of introns.

The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolish the binding of the A1 protein as well as of an anti-Sm reactive component(s) to this region. The A1, C and D proteins do not bind efficiently to fragments of either bacterial RNA or the intronless spliced product (mRNA). The binding of these proteins at the 3' end of the intron does not require addition to the extract of exogenous ATP, but remains after ATP addition. These findings demonstrate that several hnRNP proteins have RNA binding specificities on pre-mRNA, and suggest a model for hnRNP particle structure and assembly.