Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.     

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.     

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.     

hnRNP proteins interact with nascent Adenovirus RNA

By using Adenovirus 2 infected HeLa cells labeled during very brief pulses of (³H)Uridine, we have shown that nascent chains of heterogenous nuclear RNA (hnRNA) were already associated with proteins to form ribonucleoprotein particles (hnRNP). It was also shown that the small Ad2 specific VA RNA was not associated with these hnRNP.     

hnRNP L is required for the translation mediated by HCV IRES

Translation of hepatitis C virus (HCV) RNA is initiated by internal loading of the ribosome into the HCV internal ribosome entry site (IRES). Previously, heterogeneous ribonucleoprotein L (hnRNP L) was shown to bind specifically to the 3′ border region of the HCV IRES and enhance HCV mRNA translation. Here, we provide evidence for the functional requirement of hnRNP L for the HCV IRES-mediated translation initiation using specific RNA aptamers. In vitro selection techniques were employed to isolate RNA aptamers against hnRNP L, which were shown to contain consensus sequences with repetitive ACAC/U. The hnRNP L-specific RNA aptamers efficiently inhibited the in vitro translation reactions mediated by the HCV IRES in rabbit reticulocyte lysates. RNA ligands with only (ACAU)5 or (AC)10 nucleotide sequences could also specifically bind to hnRNP L, and specifically and effectively impeded in vitro translation reactions controlled by the HCV IRES. Importantly, the hnRNP L-specific RNA aptamers inhibited the HCV IRES function in cells in a dose-dependent manner, and the aptamer-mediated inhibition of the HCV IRES was considerably relieved by the addition of hnRNP L-expressing vector. These results strongly demonstrate the functional requirement of cellular hnRNP L for the HCV IRES activity.     

40S hnRNP particles are a novel class of nuclear biomolecular condensates

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.     

hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs.

Many hnRNP proteins and snRNPs interact with hnRNA in the nucleus of eukaryotic cells and affect the fate of hnRNA and its processing into mRNA. There are at least 20 abundant proteins in vertebrate cell hnRNP complexes and their structure and arrangement on specific hnRNAs is likely to be important for the processing of pre-mRNAs. hnRNP I, a basic protein of ca. 58,000 daltons by SDS-PAGE, is one of the abundant hnRNA-binding proteins. Monoclonal antibodies to hnRNP I were produced and full length cDNA clones for hnRNP I were isolated and sequenced. The sequence of hnRNP I (59,632 daltons and pI 9.86) demonstrates that it is identical to the previously described polypyrimidine tract-binding protein (PTB) and shows that it is highly related to hnRNP L. The sequences of these two proteins, I and L, define a new family of hnRNP proteins within the large superfamily of the RNP consensus RNA-binding proteins. Here we describe experiments which reveal new and unique properties on the association of hnRNP I/PTB with hnRNP complexes and on its cellular localization. Micrococcal nuclease digestions show that hnRNP I, along with hnRNP S and P, is released from hnRNP complexes by nuclease digestion more readily than most other hnRNP proteins. This nuclease hypersensitivity suggests that hnRNP I is bound to hnRNA regions that are particularly exposed in the complexes. Immunofluorescence microscopy shows that hnRNP I is found in the nucleoplasm but in addition high concentrations are detected in a discrete perinucleolar structure. Thus, the PTB is one of the major proteins that bind pre-mRNAs; it is bound to nuclease-hypersensitive regions of the hnRNA-protein complexes and shows a novel pattern of nuclear localization.     

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].     

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.     

U1 SnRNP association with HnRNP involves an initial non-specific splice-site independent interaction of U1 SnRNP protein with HnRNA

Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix

The sera of patients with mixed connective tissue disease (MCTD) have high titers of antibodies directed against nuclear U1-ribonucleoprotein (U1-RNP). This antigen is easily extracted from nuclear preparations with physiologic saline and from tissue sections with 0.1 HCl, leaving the nucleic acids and nuclear matrix behind. When U1-RNP is extracted from HEp-2 cells with 0.1 N HCl, the sera of 32/32 patients with MCTD react with another antigen that is exposed by the extraction procedure. This antigen is not destroyed by trypsin and deoxyribonuclease 1 treatment but is sensitive to both purified ribonuclease A and purified micrococcal nuclease. Absorption studies showed that the antibody reacting with this antigen cannot be absorbed by sheep red blood cells coated with extracts of rabbit thymus that contain U1-RNP. Radioimmunoassay showed that the reaction of the unadsorbed antibody was with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) and not with transfer RNA or ribosomal RNA. The hnRNP/RNA antigen is demonstrated as discrete particles in the internucleolar chromatin of interphase cells, but in metaphase cells the antigen is diffusely dispersed. The distribution, solubility, and biochemical characteristics suggest that the antigenic moiety is part of the nuclear matrix. Therefore, MCTD sera contain antibodies that react with at least two species of nuclear RNP: small nuclear RNP (snRNP), as described by others, and a high m.w. hnRNP/RNA bound to the nuclear matrix.     

The polyadenylic sequences in the ribonucleic acid of the fern Anemia phyllitidis

The vegetative prothalli (1–3 weeks old) of Anemia were incubated for 24 h in [¹⁴C]adenine. The RNA was phenol extracted from whole cells and the poly (A) sequences were isolated by nuclease digestion followed by poly (U)-sepharose chromatography. About 2–3% of the total radioactivity was retained on the column. The base composition of the nuclease resistant RNA was: C, 1.4; G, 3.6; A, 93.3; and U, 1.7. It is concluded that Anemia RNA contains poly adenylate sequences.Part of a post-doctoral work. Fellowship awarded by Alexander von Humboldt-Stiftung, Federal Republic of Germany     

Nuclear actin is associated with a specific subset of hnRNP A/B-type proteins

Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA–protein and protein–protein cross-linking experiments showed that DBP40 interacts with poly(A)⁺ RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.