An antiparallel four-helix bundle orients the high-affinity RNA binding sites in hnRNP C: a mechanism for RNA chaperonin activity.

Previous studies have shown that the C protein of 40 S hnRNP complexes contains a leucine-zipper domain, residues 180-207, and that a 40 residue highly basic domain, immediately preceding the zipper, is responsible for almost all of the free energy of RNA binding to C protein. Because this domain arrangement is like that seen in the bZIP transcription factors it has been termed the bZIP-like-motif or bZLM. We report here that the zipper domain drives C protein oligomerization through its spontaneous assembly into an anti-parallel four-helix bundle approximately 50 A in length. The anti-parallel nature of the four-helix bundle positions the tetramer's four high-affinity RNA binding domains at opposing ends of a rigid core formed by the helix bundle. This domain topology is ideally suited to accommodate and direct a double wrapping of RNA around the tetramer and is fully consistent with C protein's ability to bind and order 230 nt lengths of pre-mRNA through a highly cooperative RNA binding mode. We have used a novel sequence-specific 13C/15N labeling strategy and multidimensional NMR spectroscopy to define the anti-parallel orientation of the four-helix bundle and its molecular dimensions. In vitro reconstitution and hydrodynamic studies on native C protein, on several C protein fragments, and on various synthetic peptides, are consistent with the proposed model and indicate that C protein's canonical RNA recognition motifs probably function in tetramer-tetramer interactions during 40 S hnRNP assembly.

The bZIP-like motif of hnRNP C directs the nuclear accumulation of pre-mRNA and lethality in yeast.

The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo. Based on these and other findings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors. We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae. Through a series of fluorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA. Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-affinity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214). Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein's salt-resistant RNA binding mode. Taken together, these findings further distinguish the bZIP-like domain as the major determinant of C protein's high-affinity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity. Finally, these findings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast. Lethality is likely due to the absence in yeast of specific mechanisms for the removal of human C protein from nascent transcripts.

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.

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 ( approximately 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.

Oligonucleotide binding specificities of the hnRNP C protein tetramer.

Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3' intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3' splice site, IVS2 of human alpha-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20were used as competitors. A 20 nt beta-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines.

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein's asymmetry, its large occluded site size, and its RNA folding activity.

Proteins C1 and C2 of heterogeneous nuclear ribonucleoprotein complexes bind RNA in a highly cooperative fashion: support for their contiguous deposition on pre-mRNA during transcription.

Proteins C1 and C2 together comprise about one-third the protein mass of mammalian core 40S heterogeneous nuclear ribonucleoprotein particles (40S hnRNP) and exist as heterotetramers of (C1)3C2. On the basis of nonequilibrium binding studies, it has been suggested that the C proteins specifically bind oligo(U)- and poly(U)-rich sequences, and preferentially associate with uridine-rich regions near the 3' termini of many introns. We describe here a more quantitative characterization of the equilibrium binding properties of native and recombinant C protein to homoribopolymers using fluorescence spectroscopy. Like C protein from HeLa cells, the recombinant proteins spontaneously oligomerize to form tetramers with the same hydrodynamic properties as native protein. Near-stoichiometric binding titrations of the fluorescent homoribopolymer polyethenoadenosine (poly[r(epsilon A)]) with recombinant (C1)4 and (C2)4 homotetramers along with competition binding assays with poly(A) and poly(C) indicate that the binding site size (n) is between 150 and 230 nucleotides. This site size range is in close agreement with that previously determined for native C protein through hydrodynamic and ultrastructural studies (approximately 230 nucleotides). (C1)4 and (C2)4 bind poly(G) with intrinsic affinities (Ki) of 10(9) M-1, which are a hundredfold higher than their affinities for poly(U). In opposition to reports that C protein does not bind poly(A) and poly(C), we find that the C proteins bind these substrates with moderate Ki, but with high cooperativity (omega). The overall affinity (K omega) for the binding of both proteins to poly(A) and poly(C) is 10-fold higher (> 10(8) but < 10(9) M-1) than their affinities for poly(U). The highly cooperative binding of C protein to these substrates provides a mechanistic basis for the distribution of C protein along the length of nucleic acid substrates.

Ultrastructural morphology of the hnRNP C protein tetramer.

The C protein tetramer is one of three heterotetramers which comprise the majority of the protein mass of mammalian 40S nuclear ribonucleoprotein particles (hnRNP particles). The events of RNA processing occur while the nascent transcripts are packaged in these structures. The C protein tetramer contains three monomers of C1 and one C2 monomer [i.e., (C1)3C2]. The tetramer's mass (129.2 kDa), approximate sedimentation coefficient (5.8S), and Stokes radius (6.2 nm) suggest that the tetramer may be either highly anisotropic or may possess an unusually large hydration shell in solution. The tetramer binds approximately 235 nucleotides of pre-mRNA. Electron microscopy of purified individual RNA-free C protein tetramers has revealed the overall morphology of this important pre-mRNA binding complex. In negatively stained preparations, the tetramer clearly displays a nonlinear, three- or four-lobed appearance with a diameter of 8.5 +/- 0.5 nm. A detailed comparison of the substructure seen in individual images suggests a tetrahedral arrangement of the four polypeptides. Rotary-shadowed images confirm the size of the tetramer observed in negatively stained preparations. This study provides the first demonstration of the overall arrangement of polypeptides in the C protein tetramer.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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].

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.

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.

Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.

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.

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.

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).

Distribution studies on polytene chromosomes using antibodies directed against hnRNP.

The distribution of nuclear ribonucleoprotein (hnRNP) particles in Drosophila polytene chromosomes has been investigated using anti-B-36 serum as a probe. The use of polytene chromosomes allows resolution at the level of the chromomere, and provides the opportunity to look for both positive and negative correlations with transcriptional activity. The antiserum was obtained using the nuclear protein B-36 from Physarum polycephalum as the immunogen. It has been shown to precipitate hnRNP particles from HeLa cells through a cross-reaction with the major 32,000- and 34,000-dalton hnRNP particle proteins. The antiserum cross-reacts with a Drosophila nuclear protein of approximately 34,000 daltons. By indirect immunofluorescence, we observed that the antiserum reacts preferentially with transcriptionally active loci of the polytene chromosomes, whereas loci previously or subsequently active do not show significant fluorescence. The overall pattern of fluorescence is very similar to that generated with anti-RNA polymerase B serum. The correlation of fluorescence and transcriptional activity observed suggests that the anti-B-36 serum is recognizing hnRNP proteins which have combined with nascent RNA molecules at the sites of transcription.

The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease.

Brief digestion of HeLa nuclei with mirococcal nuclease releases monomer hnRNP particles as well as monomer and polynucleosomes. Sucrose gradient analysis of the nuclease released material reveals a series of small A260 peaks overlapping a more predominant peak in the 40S region of the gradient. Analysis of the proteins, DNa, and RNA in successive gradient fractions has confirmed that the smaller peaks are monomer and polynucleosomes, and that the larger peak is 40S hnRNP. Like 40S particles isolated by low salt extraction or by sonication, the nuclease released particles are composed of rapidly labeled RNA associated with a group of non-histone proteins the most predominant of which are the 32,000-44,000 MW proteins previously identified as core hnRNP proteins. These results provide further evidence that 40S hnRNP particles exist as discrete structural components of larger in vivo ribonucleoprotein complexes.