The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization.

More than 20 different heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with pre-mRNAs in the nucleus of mammalian cells and these proteins appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. The arrangement of hnRNP proteins on pre-mRNAs is likely to be unique for each RNA and may be determined by the different RNA-binding preferences of each of these proteins. hnRNP F (M(r) = 53 kD, pI = 6.1) and hnRNP H (M(r) = 56 kD, pI = 6.7-7.1) are abundant components of immunopurified hnRNP complexes and they have distinct nucleic acid binding properties. Unlike other hnRNP proteins which display a varying range of affinities for different ribonucleotidehomopolymers and ssDNA, hnRNP F and hnRNP H bind only to poly(rG) in vitro. hnRNP F and hnRNP H were purified from HeLa cells by poly(rG) affinity chromatography and oligonucleotides derived from peptide sequences were used to isolate a cDNA encoding hnRNP F. The predicted amino acid sequence of hnRNP F revealed a novel protein with three repeated domains related to the RNP consensus sequence RNA-binding domain. Monoclonal antibodies produced against bacterially expressed hnRNP F were specific for both hnRNP F and hnRNP H and recognized related proteins in divergent organisms, including in the yeast Saccharomyces cerevisiae. hnRNP F and hnRNP H are thus highly related immunologically and they share identical peptides. Interestingly, immunofluorescence microscopy revealed that hnRNP F and hnRNP H are concentrated in discrete regions of the nucleoplasm, in contrast to the general nucleoplasmic distribution of previously characterized hnRNP proteins. The unique RNA-binding properties, amino acid sequence and distinct intranuclear localization of hnRNP F and hnRNP H make them novel hnRNP proteins that are likely to be important for the processing of RNAs containing guanosine-rich sequences.     

Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single- stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.     

HnRNP proteins and the nuclear export of mRNA

Pre-mRNAs associate in the nucleus with specific RNA-binding proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. The hnRNP proteins participate directly or indirectly in the processing of pre-mRNAs into mature mRNAs. Recent studies have shown that some hnRNP proteins shuttle continuously between the nucleus and the cytoplasm. The export of shuttling hnRNP proteins from the nucleus is mediated by specific nuclear export sequences (NESs) within the proteins. In addition, shuttling hnRNP proteins appear to remain bound to exported mRNAs in transit through nuclear pores. As discussed in this review, the picture that is emerging is that nuclear export of mRNAs is mediated by the export of NES-containing hnRNP proteins to which they are bound.     

hnRNA and its attachment to a nuclear protein matrix

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.     

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.     

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.     

RNA annealing activities in HeLa nuclei.

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.