Heterogeneous Nuclear Ribonucleoprotein Particles Research Articles

An Ultrastructural Characterization of in Vitro-Assembled hnRNP C Protein-RNA Complexes

In mammalian cells approximately 700 nucleotide lengths of pre-mRNA are packaged during transcription by a unique group of abundant nuclear proteins to form a repeating array of regular ribonucleoprotein complexes termed 30-40S heterogeneous nuclear ribonucleoprotein particles (hnRNP particles). We have used electron microscopy to examine complexes that form when in vitro-transcribed RNA is bound by one of the purified native core-particle proteins which comprise the 40S monoparticle (the C protein tetramer). Negatively stained images of the C protein tetramer bound to particle-length RNA (700 nt) demonstrate that three tetramers bind each RNA molecule to form a stable closed triangular complex. The triangular complexes have an isosceles shape with a base of 18.0 nm and sides of 23.0 nm. When RNA molecules of 230 nt are used as substrates single tetramers bind to form complexes that appear as small rounded structures with an average diameter of 9.7 nm. Twice this length of RNA (456 nt) supports the assembly of mostly bilobed complexes that are 20.4 nm long and 11.8 nm wide. Images of the C protein-RNA complexes which assemble on 1400-nucleotide lengths of RNA (two particle lengths of RNA) clearly show complexes composed of two triangles while three-triangle complexes are seen when 2100-nt lengths of RNA are used as the assembly substrate. These ultrastructural results demonstrate that groups of three C protein tetramers combine with the length of RNA packaged in monoparticles to form a discreet triad structure. This interaction establishes a structural basis for the RNA length requirement in monoparticle assembly and a mechanism for the packaging of nascent transcripts into a repeating array of regular structures.

Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver.

A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.

Chicken MAR binding protein p120 is identical to human heterogeneous nuclear ribonucleoprotein (hnRNP) U.

We have previously identified two proteins from chicken oviduct nuclei that specifically bind to matrix/scaffold attachment regions (MARs/SARs). Here one of these proteins, named p120 due to its apparent molecular weight, is purified to near homogeneity and shown to be identical to a previously described component of heterogeneous nuclear ribonucleoprotein particles, hnRNP U, on the basis of amino acid sequence analysis of tryptic peptides. p120 binds to multiple MAR fragments provided they have a minimal length of approximately 700 bp. Binding of MAR fragments is specifically competed by homoribopolymers poly(G) and poly(I), which form four-stranded structures. Our results suggest that p120/hnRNP U may serve a dual function, first as a component of hnRNP particles, and second as an element in the higher-order organization of chromatin.

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.

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.

In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism

We describe the nuclear organization of pre-mRNA processing components in HeLa cells upon adenovirus 2 infection and their relationship to the localization of viral RNA sequences. We observe a redistribution of cellular splicing factors as well as RNA polymerase II and heterogeneous nuclear ribonucleoprotein particle proteins to sites of viral RNA transcription. Similar results were obtained in cells transiently transfected with a plasmid containing a portion of the β-tropomyosin gene. Our findings demonstrate a very close association between RNA transcripts and transcription and pre-mRNA splicing factors, suggesting that these processes are both temporally and spatially linked in the cell nucleus. Furthermore, these data suggest a recruiting mechanism that regulates the localization of transcription and splicing factors in response to the initiation of active transcription.

Enzymatic methylation of heterogeneous nuclear ribonucleoprotein in isolated liver nuclei

Protein N-methyltransferase activity has been studied in the rat liver nuclei, using recombinant heterogeneous nuclear ribonucleoprotein particle protein A1 and histone as the methyl acceptors. The hydrolysates of these two enzymatically [methyl-3H]-labeled proteins, however, yielded different patterns of methylated amino acids on HPLC analysis: NG-monomethylarginine (92%) and NG-NG-dimethyl (asymmetric) arginine (6.5%) were the major methylated amino acids identified in the protein A1, whereas epsilon-N-methylated lysine derivatives constituted a predominant portion (71%) of the methylated amino acids in histone. When liver extracts isolated from rats fed a methyl deficient diet were assayed, the methyl accepting activity of protein A1 increased 64% over the control (rats fed normal diet), while that of histone increased 260%. Partial hepatectomy induced a 7.9-fold and 2.3-fold increase in the protein A1 methylase activity after 24 and 48 h of regeneration, respectively. These results, together with the fact that myelin basic protein-specific protein methylase I does not significantly methylate protein A1, indicate the presence of an enzyme in the rat liver nuclei which methylates the protein A1.

Sex determination in loggerhead turtles: differential expression of two hnRNP proteins

Sex determination in the loggerhead turtle, Caretta caretta, is controlled by incubation temperature during a critical period of embryogenesis. As heat-shock gene expression is temperature-dependent and has been shown to be associated with early developmental regulation in several organisms, we studied the constitutive expression of hsp70 and hsp90 in embryonic brain and urinogenital tissues to see if these proteins are differentially expressed during the sex-determining period in embryos incubated at male- (26 degrees C) and female- (32 degrees C) determining temperatures. The level of expression of hsp70 and hsp90, as determined from monoclonal antibody staining, is similar in both sexes during the sex-determining period. However, AC88, a monoclonal antibody that identifies hsp90 in several systems, recognised two additional protein bands (Mr 42 and 46 x 10(3)), which are differentially expressed in the urinogenital tissue of developing male and female embryos during the sex-determining period. While the 42K and 46K proteins appear in the urinogenital tissue of developing female (32 degrees C) embryos until stage 25, they are not expressed in the male (26 degrees C) urinogenital system after stage 24. Subsequent experiments have identified both turtle proteins as heterogeneous nuclear ribonucleoprotein particles (hnRNPs). As several hnRNP proteins have specific RNA-binding sites and are involved in mRNA processing reactions, the 46K protein may mediate post-transcriptional control of specific RNA transcripts required for sexual differentiation in C. caretta.

A single-stranded nucleic acid binding sequence common to the heterogeneous nuclear ribonucleoparticle protein A1 and murine recombinant virus gp70.

We have used an antisynthetic peptide antiserum to a murine recombinant virus gp70 to probe normal mouse tissues for immunologically related proteins. In addition to cognate gp70s, this antiserum reacts with the heterogeneous nuclear ribonucleoparticle protein A1 by virtue of a 5-amino acid epitope, PRNQG. Further structural similarity is evident both 5' and 3' of this epitope. Since the function of the heterogeneous nuclear ribonucleoprotein particles in the cell is to aid in the stabilization and processing of newly synthesized RNA, we have investigated whether this retroviral sequence exhibits any nucleic acid-binding properties by the same criteria established for the identification of heterogeneous nuclear ribonucleoprotein particles. Analysis of the peptide in a poly(eA) binding assay shows this retroviral sequence to bind with high affinity to single-stranded nucleic acid. This binding occurs in a salt-sensitive manner characteristic of single-stranded nucleic acid-binding proteins. Flanking peptides not containing this sequence generated from either the A1 or gp70 show no ability to bind single-stranded nucleic acids by this assay.

The Drosophila Hrb98DE Locus Encodes Four Protein Isoforms Homologous to the A1 Protein of Mammalian Heterogeneous Nuclear Ribonucleoprotein Complexes

The Drosophila Hrb98DE locus encodes proteins that are highly homologous to the mammalian A1 protein, a major component of heterogeneous nuclear ribonucleoprotein (RNP) particles. The Hrb98DE locus is transcribed throughout development, with the highest transcript levels found in ovaries, early embryos, and pupae. Eight different transcripts are produced by the use of combinations of alternative promoters, exons, and splice acceptor sites; the various species are not all equally abundant. The 3'-most exon is unusual in that it is completely noncoding. These transcripts can potentially generate four protein isoforms that differ in their N-terminal 16 to 21 amino acids but are identical in the remainder of the protein, including the RNP consensus motif domain and the glycine-rich domain characteristic of the mammalian A1 protein. We suggest that these sequence differences could affect the affinities of the proteins for RNA or other protein components of heterogeneous nuclear RNP complexes, leading to differences in function.

The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes.

The Drosophila Hrb98DE locus encodes proteins that are highly homologous to the mammalian A1 protein, a major component of heterogeneous nuclear ribonucleoprotein (RNP) particles. The Hrb98DE locus is transcribed throughout development, with the highest transcript levels found in ovaries, early embryos, and pupae. Eight different transcripts are produced by the use of combinations of alternative promoters, exons, and splice acceptor sites; the various species are not all equally abundant. The 3'-most exon is unusual in that it is completely noncoding. These transcripts can potentially generate four protein isoforms that differ in their N-terminal 16 to 21 amino acids but are identical in the remainder of the protein, including the RNP consensus motif domain and the glycine-rich domain characteristic of the mammalian A1 protein. We suggest that these sequence differences could affect the affinities of the proteins for RNA or other protein components of heterogeneous nuclear RNP complexes, leading to differences in function.

Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells.

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the 'core' proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.

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.

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.

Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles.

Partial acid cleavage, comparative HPLC tryptic peptide mapping and amino acid sequencing of the C1 and C2 proteins of HeLa heterogeneous nuclear ribonucleoprotein (hnRNP) particles demonstrate that proteins C1 and C2 differ in primary structure by the presence of a 13 amino acid insert sequence in C2. This C2 insert sequence occurs after either glycine 106 or serine 107 in C1. The additional 13 amino acids that are present in C2 account for the observed molecular weight difference between the C1 and C2 hnRNP proteins on SDS polyacrylamide gel electrophoresis. Because C1 and C2 appear identical except for the 13 residue insert and because the 3' and 5' untranslated regions of the corresponding mRNAs also appear to be the same (Swanson et al., Mol. Cell. Biol. 7: 1731-1739), it is possible that both polypeptides are produced from a single transcription unit through an alternative splicing mechanism.

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation.

Abstract Heterogeneous nuclear ribonucleoprotein (hnRNP) particles, the structures that package hnRNA, are one of the major constituents of the nucleus. Recent work has led to the immunopurification of hnRNP particles and the identification of their proteins, and demonstrated a role for hnRNP proteins in mRNA splicing. The molecular cloning and sequencing of cDNAs for RNP proteins made possible the discovery of a conserved RNA-binding domain and a RNP consensus sequence.

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin- and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.

Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.

A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.