Correlated alternative side chain conformations in the RNA-recognition motif of heterogeneous nuclear ribonucleoprotein A1

The RNA-recognition motif (RRM) is a common and evolutionarily conserved RNA-binding module. Crystallographic and solution structural studies have shown that RRMs adopt a compact α/β structure, in which four antiparallel β-strands form the major RNA-binding surface. Conserved aromatic residues in the RRM are located on the surface of the β-sheet and are important for RNA binding. To further our understanding of the structural basis of RRM-nucleic acid interaction, we carried out a high resolution analysis of UP1, the N-terminal, two-RRM domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), whose structure was previously solved at 1.75–1.9 Å resolution. The two RRMs of hnRNP A1 are closely related but have distinct functions in regulating alternative pre-mRNA splice site selection. Our present 1.1 Å resolution crystal structure reveals that two conserved solvent-exposed phenylalanines in the first RRM have alternative side chain conformations. These conformations are spatially correlated, as the individual amino acids cannot adopt each of the observed conformations independently. These phenylalanines are critical for nucleic acid binding and the observed alternative side chain conformations may serve as a mechanism for regulating nucleic acid binding by RRM-containing proteins.     

Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts with heterogeneous nuclear ribonucleoprotein L

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the principal pre-mRNA-binding proteins found in human cells. The hnRNP L protein is fairly abundant. However, it is not restricted to the nucleus, and instead shuttles between the nucleus and the cytoplasm. It is composed of 558 amino acid residues and harbours four loosely conserved RNP-consensus RNA-binding domains. In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study, we determined that hnRNP L interacts specifically with the hnRNP D/AUF1 in the yeast two-hybrid system. This interaction was verified via an in vitro pull-down assay.     

Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts with heterogeneous nuclear ribonucleoprotein L

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the principal pre-mRNA-binding proteins found in human cells. The hnRNP L protein is fairly abundant. However,it is not restricted to the nucleus, and instead shuttles between the nucleus and the cytoplasm. It is composed of 558 amino acid residues and harbours four loosely conserved RNP-consensus RNA-binding domains.In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study,we determined that hnRNP L interacts specifically with the hnRNP D/AUF1 in the yeast two-hybrid system. This interaction was verified via an in vitro pull-down assay.     

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.     

Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly

Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.     

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.     

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA, telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of mRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem betaalpha betabeta alphabeta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.     

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.     

Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1.

In order to improve the activity of hammerhead ribozymes in vivo, we have analyzed the effect of several prototypical RNA binding proteins on the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp32), hnRNP A1 (A1) and the nucleocapsid protein of HIV-1 (NCp7). We show that, while gp32 has no effect on the cleavage reaction, A1 and NCp7 affect different steps of the reaction. Moreover, some of these effects depend upon the ribozyme-substrate hybrid length. A1 and NCp7 inhibit the reaction of the least stable ribozyme-substrate complexes, which have 12 bp of duplex. NCp7, but not A1, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), while cleavage of complexes having shorter duplexes (13 or 14 bp) is not affected. NCp7 and A1 enhance the turnover of ribozymes by increasing the rate of product dissociation, but only when both cleavage products are bound with < or = 7 bp. A1 and NCp7 enhance ribozyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of A1 or NCp7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Interestingly, this duplex length (14 bases, or 7 on each side of the cleavage site) is identical for A1 and NCp7. Since A1 is thought to interact with most, if not all mRNAs in vivo, it may enhance the intracellular activity of ribozymes targeted against any mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The heterogeneous nuclear ribonucleoprotein K is important for Herpes simplex virus-1 propagation

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is an evolutionarily conserved protein with roles in signal transduction and gene expression. An impact of hnRNP K on the life cycle of a broad range of viral pathogens was reported while functional data for herpesviruses were lacking. In this study we show that hnRNP K is important for Herpes simplex virus 1 egress. In absence of hnRNP K, viral entry, gene expression, viral DNA replication, and maturation of nuclear particles appear normal whereas release of infectious virions to the extracellular space was significantly affected. Our results indicate that hnRNP K has an impact on a late step of herpesviral propagation making it a potential antiviral target.     

Molecular characterization of a mouse heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue-specific expression

The human DNA- and RNA-binding protein JKTBP is a new member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis. We cloned and characterized a mouse homolog and studied its expression in mouse tissues. The cDNA encoded a 301-residue polypeptide. There is only a single amino acid difference between the mouse and human sequences. Northern blotting indicated ubiquitous but varied expressions of approximately 1.4 and 2.8kb mRNAs in various tissues. Immunoblotting indicated that the amounts of protein of about 38kDa were higher in the brain and testis than in other tissues. An additional protein of about 53kDa was found in the brain and testis. Germ cell-deficient W/W(v) mutant mice and aged mice had the reduced amounts of JKTBP in the testes. Immunohistochemical staining indicated cell type-specific expression of JKTBP in tissues: neurons and spermatocytes displayed strong signal intensities. The signals were confined to the nucleus. The amount of 38kDa JKTBP was estimated to be approximately 1.3x10(7) molecules per HL-60 cell. These results indicate that JKTBP is an abundant, highly conserved nuclear protein.     

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis

The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.     

Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.     

Interaction and stimulation of human FEN-1 nuclease activities by heterogeneous nuclear ribonucleoprotein A1 in alpha-segment processing during Okazaki fragment maturation

High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.     

Limited nuclease digestion of heterogeneous nuclear ribonucleoprotein in nuclei.

We have used limited nuclease digestion of nuclei to probe the structure of nuclear ribonucleoprotein (nRNP). Analysis of [³H]uridine-labeled heterogeneous nuclear RNA isolated from nuclease digested nuclei revealed preferential generation of discrete bands of RNA ranging in size from 1.5 × 10⁵ to 6 × 10⁵ daltons. The nuclease digestion pattern of nRNP differed from the nuclease digestion pattern obtained with chromatin in that the RNA bands generated in these experiments were transient, appearing only early in the course of digestion, and no stable nRNP monomer size was evident. Therefore, although nRNP may be organized in a regular configuration, nRNP structure differs considerably from the repeating subunit structure of chromatin.