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.     

Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex

In previous studies, a lectin designated as carbohydrate binding protein 35 (CBP35) was identified in the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts. In the present study, we observed that treatment of Triton X-100 permeabilized 3T3 cells with ribonuclease A released CBP35 from the nuclei, while parallel treatment with deoxyribonuclease I failed to do so. This conclusion was based on (a) immunofluorescence analysis of the nuclear residue after detergent and enzymatic treatments and (b) immunoblotting analysis of the supernatant fraction produced by these treatments. These results indicate that CBP35 may be associated with the ribonucleoprotein elements of the 3T3 cell nuclei. In corroboration with this conclusion, fractionation of the nucleoplasm derived from 3T3 cells on a cesium sulfate gradient (1.25-1.75 g/mL) localized CBP35 in fractions with densities of 1.30-1.32 g/mL, corresponding to the range of densities reported for heterogeneous nuclear ribonucleoprotein complex (hnRNP). Conversely, when nucleoplasm was fractionated on an affinity column of Sepharose derivatized with N-(epsilon-aminocaproyl)-D-galactosamine, the bound and eluted fraction contained RNA, as well as a set of polypeptides whose molecular weights matched those reported for the core particle of hnRNP. One of these polypeptides was identified as CBP35. These results suggest that CBP35 is a component of hnRNP.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

Extensive deletions in the Q region of the mouse major histocompatibility complex

By use of Southern blot analyses and low copy number probes, the fine structure of the Q region of the mouse major histocompatibility complex was studied in more detail. With a probe recognizing the even-numbered genes Q4, Q6, and Q8, it was evident that Q4 and/or the regions flanking Q4 are polymorphic, whereas Q6 and Q8, and their flanking regions are nonpolymorphic. Perhaps the most noteworthy finding is that at least two strain haplotypes, H-2 ^k and H-2 ^f, possessed extensive deletions in the Q region. The most striking deletion was found in the H-2 ^f haplotype, where the QI through Q9 genes appear to be missing. Because of these extensive deletions the functional importance of the Q region is questioned.     

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.     

Recombination in the class III region of the mouse major histocompatibility complex

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped combiantional break-points of 79 Ab: H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order Eb-61.11-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were cofiend within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotsopt within the Int3:Tnx interval.     

Gene duplications in the TL region of the mouse major histocompatibility complex

We have isolated a class I gene from the TL region of the A/J mouse. The gene, T2A, is a homologue of the C57BL/10 mouse gene T2. In the process of mapping this gene we screened a number of BALB/c class I cosmid clusters with a T2A flanking probe. Several of the hybridizing clusters were found to contain identical DNA segments and could therefore be linked together into one single BALB/c TL region which appears to be identical to the TL region of the C57BL/10 mouse. However, two of the hybridizing clusters do not overlap with the C57BL/10 TL region. It appears that these two clusters represent a partial duplication of the TL region in the BALB/c mouse.     

A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2Kb MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)-reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptide-specific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR+ spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2Kb. This fusion protein molecule may be useful to study peptide-MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens.     

A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.     

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.     

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.     

New genes in the class II region of the human major histocompatibility complex

A detailed map of the class II region of the human major histocompatibility complex has been constructed by pulsed-field gel electrophoresis. This map revealed clusters of sites for enzymes that cut preferentially in unmethylated CpG-rich DNA often found at the 5' ends of genes. Three of these clusters have been cloned by cosmid walking and chromosome jumping. Analysis of the clones encompassing these regions through the use of zoo blots, Northern blots, and cDNA libraries resulted in the discovery of four novel genes. The D6S111E and D6S112E genes are centromeric to the HLA-DPB2 gene, while D6S113E and D6S114E are between HLA-DNA and HLA-DOB. Preliminary characterization of the new genes indicates that they are unrelated to the class II genes themselves, although D6S114E expression, like class II expression, is inducible with interferon. In addition, the HLA-DNA gene has been accurately positioned and oriented for the first time.     

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.