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PubMed1983

U1 small nuclear ribonucleoprotein studied by in vitro assembly.

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

Antibody Specificity↗

PubMed1985

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA.

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).

Animals↗

PubMed2001

Molecular dynamics and thermodynamics of protein-RNA interactions: mutation of a conserved aromatic residue modifies stacking interactions and structural adaptation in the U1A-stem loop 2 RNA complex.

Molecular dynamics (MD) simulations and free energy component analysis have been performed to evaluate the molecular origins of the 5.5 kcal/mol destabilization of the complex formed between the N-terminal RNP domain of U1A and stem loop 2 of U1 snRNA upon mutation of a conserved aromatic residue, Phe56, to Ala. MD simulations, including counterions and water, have been carried out on the wild type and Phe56Ala peptide-stem loop 2 RNA complexes, the free wild type and Phe56Ala peptides, and the free stem loop 2 RNA. The MD structure of the Phe56Ala-stem loop 2 complex is similar to that of the wild type complex except the stacking interaction between Phe56 and A6 of stem loop 2 is absent and loop 3 of the peptide is more dynamic. However, the MD simulations predict large changes in the structure and dynamics of helix C and increased dynamic range of loop 3 for the free Phe56Ala peptide compared to the wild type peptide. Since helix C and loop 3 are highly variable regions of RNP domains, this indicates that a significant contribution to the reduced affinity of the Phe56Ala peptide for RNA results from cooperation between highly conserved and highly variable regions of the RNP domain of U1A. Surprisingly, these structural effects, which are manifested as cooperative free energy changes, occur in the free peptide, rather than in the complex, and are revealed only by study of both the initial and final states of the complexation process. Free energy component analysis correctly accounts for the destabilization of the Phe56Ala-stem loop 2 complex, and indicates that approximately 80% of the destabilization is due to the loss of the stacking interaction and approximately 20% is due to differences in U1A adaptation.

Mutation↗

PubMed1988

Prosomes, small cytoplasmic RNP particles, contain glycoproteins.

Prosomes, ubiquitous small ribonucleoprotein complexes, were isolated from the cytoplasm of erythropoietic mouse cells induced by Friend leucemia virus. We present evidence that some of the prosomal proteins are glycosylated. Specific reactions with the biotinylated lectins concanavalin agglutinin (Con A), Solanum tuberosum agglutinin (STA) and Limulus polyphemus agglutinin (LPA) indicate that the carbohydrate moieties contain N-acetylneuraminic acid, N-acetylglucosamine and mannosyl- or glucosyl-residues. Glycosylation of prosomal proteins could explain the resistance of prosomes to proteinase K digestion.

Animals↗

PubMed1977

[Ribonucleoproteins containing heterogeneous nuclear and messenger cytoplasmic RNA. Characteristics, structure and relations (author's transl)].

Following the study of Spirin, many authors have shown that cytoplasmic messenger RNA and heterogeneous nuclear RNA are complexed with specific proteins to form ribonucleoprotein particles (RNP). These RNP are heterogeneous in size and present a high protein to RNA ratio. Different observations suggest a polymeric structure for nuclear ribonucleoproteins but their protein composition is more clearly complex than that of cytoplasmic ribonucleoproteins. If we accept the following processing: heterogeneous nuclear RNP leads to free cytoplasmic RNP leads to polysomic RNP, the question arises as to what extent proteins originally present in nuclear RNP are conserved. Proteins more or less tightly bound to RNA have different roles: packing and protection of RNA, regulation during gene expression processing.

Animals↗

PubMed1985

Characterization of the RNA binding properties of transcription factor IIIA of Xenopus laevis oocytes.

A nitrocellulose filter binding assay has been developed to study the interaction of Xenopus transcription factor IIIA with 5S RNA. The protein binds Xenopus oocyte 5S RNA with an association constant of 1.4 X 10(9) M-1 at 0.1 M salt, pH 7.5 at 20 degrees C. TF IIIA binds wheat germ 5S RNA with a two-fold higher affinity, E. coli 5S RNA with a four-fold weaker affinity, and has a barely detectable interaction with yeast tRNAphe. The preference for binding eukaryotic 5S RNA is enhanced in competition assays. The homologous reconstituted complex contains one molecule each of protein and 5S RNA and is indistinguishable from native 7S RNP in mobility on non-denaturing polyacrylamide gels. The conformation of the RNA in reconstituted particles is identical to the conformation of RNA in native 7S RNP. Further analysis of the homologous interaction reveals that complex formation is a favoured both by enthalpy and entropy. The 5S RNA binding activity has a broad pH optimum spanning pH 6.0 to pH 8.0. Determination of the salt dependence of Ka reveals that as many as 5 lysine-phosphate type ionic bonds may be formed in the homologous complex. Approximately 68% of the free energy of complex formation is contributed by non-electrostatic interactions between TF IIIA and Xenopus 5S RNA.

Animals↗

PubMedDate not supplied

[Reversible dissociation of influenza virus ribonucleoprotein].

Dissociation of influenza virus RNP under the effect of salt was studied. Separation of RNA and protein components of influenza virus RNP was shown to occur in a linear 15-30% sucrose concentration gradient containing 1.1 M NaCl. Upon RNP dissociation, protein-protein interactions between individual molecules of the structural protein were retained. The sedimentation coefficient of the protein component was 52S. The possibility of reassociation of the RNA-protein complex was studied. More complete reassociation was observed to occur in the presence of 0.1 M NaCl. The resulting RNA-protein complex morphologically is similar to the native RNP of influenza virus.

Animals↗

PubMed1984

Intracellular site of U1 small nuclear RNA processing and ribonucleoprotein assembly.

We have investigated the intracellular site and posttranscriptional immediacy of U1 small nuclear RNA processing and ribonucleoprotein (RNP) assembly in HeLa cells. After 30 or 45 min of labeling with [3H]uridine, a large amount of U1-related RNA radioactivity in the cytoplasm was found by using either hypotonic or isotonic homogenization buffers. The pulse-labeled cytoplasmic U1 RNA was resolved as a ladder of closely spaced bands running just behind mature-size U1 (165 nucleotides) on RNA sequencing gels, corresponding to a series of molecules between one and at least eight nucleotides longer than mature U1. They were further identified as U1 RNA sequences by gel blot hybridization with cloned U1 DNA. The ladder of cytoplasmic U1 RNA bands reacted with both RNP and Sm autoimmune sera and with a monoclonal Sm antibody, indicating a cytoplasmic assembly of these U1 RNA-related molecules into complexes containing the same antigens as nuclear U1 RNP particles. The cytoplasmic molecules behave as precursors to mature nuclear U1 RNA in both pulse-chase and continuous labeling experiments. While not excluding earlier or subsequent nuclear stages, these results suggest that the cytoplasm is a site of significant U1 RNA processing and RNP assembly. This raises the possibility that nuclear-transcribed eucaryotic RNAs are always processed in the cell compartment other than that in which they ultimately function, which suggests a set of precise signals regulating RNA and ribonucleoprotein traffic between nucleus and cytoplasm.

Autoantibodies↗

PubMed2004

Hypophosphorylated ASF/SF2 binds TAP and is present in messenger ribonucleoproteins.

Serine/arginine-rich proteins (SR proteins) function in precursor mRNA (pre-mRNA) splicing and may also act as adaptors for mRNA export. SR proteins are dynamically phosphorylated in their RS domain, and differential phosphorylation modulates their splicing activity and subcellular localization. In this study, we investigated the influence of phosphorylation on the function of SR proteins in events occurring during mRNA maturation. Immunoprecipitation experiments showed that the mRNA export receptor TAP associates preferentially with the hypophosphorylated form of shuttling SR proteins, including ASF/SF2. Overexpression of ASF induced subnuclear relocalization of TAP to SR protein-enriched nuclear speckles, suggesting their interaction in vivo. Moreover, the ASF found in a nucleoplasmic fraction rich in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes is hyperphosphorylated, whereas mature messenger RNP (mRNP)-bound ASF is hypophosphorylated. Therefore, hypophosphorylation of ASF in mRNPs coincides with its higher affinity for TAP, suggesting that dephosphorylation of ASF promotes both its incorporation into mRNPs and recruitment of TAP for mRNA export. Thus, the phosphorylation state of RS domains may modulate the function of mammalian shuttling SR proteins during mRNA maturation or export.

Base Sequence↗

PubMed2005

Stepwise functionalization of ribonucleopeptide complexes to receptors and sensors.

We describe here stepwise functionalization strategies of ribonucleopeptide complexes to receptors and sensors. A structurally well-defined complex of RNA and peptide (ribonucleopeptide: RNP) was constructed by a structure-based design. The first step optimizes an ATP-binding characteristic of an RNP receptor based on an in vitro selection of an RNP library generated by introducing randomized nucleotide sequences in the RNA subunit. In the second step, the RNP receptor from the first step was functionalized to an RNP receptor for nicotinamide adenine dinucleotide (NAD+) or to ATP sensors by using respective modifications of the peptide subunit.

Adenosine Triphosphate↗

PubMed1986

[Comparative evaluation of the sensitivity of immunofluorescence methods, immunoenzyme analysis and the lectin test for the rapid diagnosis of influenza].

Comparative study of the sensitivities of immunofluorescent microscopy (IFM), enzyme immunoassay, (EIA), and lectin test (LT) in the detection of influenza virus antigen in nasopharyngeal washings from patients with influenza, acute respiratory diseases, pneumonia, and laryngitis has been carried out. EIA modification (used in this study) based on the detection of a complex of viral core proteins (M + RNP) has been shown to be no less sensitive than IFM and suitable for use in the rapid diagnosis of influenza. It can be used in combination with other methods. The optimum time for collecting the washings off is day 2 from the disease onset for analysis by EIA technique and day 4 for LT.

Adolescent↗

PubMed1980

Ultrastructural variations following use of several weakly protic alcohols as dehydration agents.

Evaluation has been made of ultrastructural variations which obtain following dehydration and infiltration of glutaraldehyde plus osmium fixed CHO cells with the weakly protic alcohols (methanol, ethanol, ethylene glycol, 1-propanol, 2-propanol, propylene glycol and glycerine), acetone alone, and with use of acetone and propylene oxide as transition solvents subsequent to dehydration with ethanol, methanol and ethylene glycol. It appears that the weakly protic alcohols, applied without transition solvent, in addition to some extraction by themselves, variously condition the components of membranes, etc, for possible further extraction by the embedment resin. Extraction by embedment consists mainy of removal of aggregate or 'corpuscular' units from regions assumed to have originally consisted of endomembrane. Relatively discrete extraction pits in membranes are noted in the cases of methanol and ethylene glycol dehydration without transition solvent; whereas use of the other alcohols such as 1-propanol and 2-propanol accentuate the visualization and staining of mitochondrial ribosomes. Cytoplasmic ribosomes stain more strongly when acetone or ethylene glycol are employed as dehydration agents, but some size (length) differences obtain. Apparently, acetone or propylene oxide transition following dehydration with ethanol or methanol (but not the glycols) assists in, or fixes, alterations of endomembrane components into the familiar 'unit membrane' orientation upon which the embedding substances have little discernible effect. Glycol dehydration and infiltration regimens (especially ethylene glycol alone and as a dehydration agent in the application of Bernhard's (1969) staining technique) result in lesser electron density of heterochromatin while accentuating RNP particles. The increased electron density of heterochromatin while accentuating RNP particles. The increased electron density of the RNP particles and the decrease in density of the heterochromatin result in improved visualization of the relationships among nucleolar RNP particles and the nuclear pore complexes.

Alcohols↗

PubMed1993

Contrasting molecular patterns of MHC class II alleles associated with the anti-Sm and anti-RNP precipitin autoantibodies in systemic lupus erythematosus.

OBJECTIVE: To find evidence of a potential genetic predisposition to the anti-Sm or anti-RNP precipitin autoantibody responses. METHODS: HLA-DR and DQ alleles determined by restriction fragment length polymorphism and/or oligotyping in 49 subjects with either anti-Sm alone or anti-RNP alone were compared with those in 139 race-matched normal control subjects and 59 race-matched lupus patients without anti-Sm and anti-RNP autoantibodies. RESULTS: Black patients with anti-Sm precipitin had increased frequencies of HLA-DR2 and the DQw6-associated DQA1*0102 (P = 0.007, odds ratio [OR] = 6.7) and DQB1*0602 (P = 0.001, OR = 9.1) chain alleles compared with normal black control subjects. Black patients with anti-RNP precipitin showed significant increases in the DQw5-associated DQA1*0101 (P = 0.03, OR = 5.5) and DQB1*0501 (P = 0.002, OR = 23.3) chain alleles compared with lupus patients without anti-Sm or RNP. While patients with anti-RNP precipitin showed an increased frequency of the DQw8-associated allele DQB1*0302 (P = 0.02, OR = 3.7) compared with normal controls, as well as an increased frequency of the DQw5-associated alleles DQA1*0101 and DQB1*0501 (P = 0.05, OR = 4.2) compared with lupus patients without anti-Sm or RNP. There were no specific HLA-DR2 or DR4 subtype associations found with either anti-Sm or RNP precipitin autoantibodies. CONCLUSION: There are distinct patterns of major histocompatibility complex class II allele associations with the anti-Sm versus the anti-RNP precipitin autoantibody responses, and HLA-DQ associations may be more primary than HLA-DR associations.

Alleles↗

PubMed1999

Genetic and physical interactions involving the yeast nuclear cap-binding complex.

Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.

Gene Deletion↗

PubMed2001

A human RNA polymerase II-containing complex associated with factors necessary for spliceosome assembly.

Transcription and splicing are coordinated processes in mammalian cells. We have used affinity chromatography with immobilized transcription elongation factor SII to purify a protein complex that contains core RNA polymerase II (RNA Pol II), the general transcription initiation factors, and several splicing factors, including the U1, U2, and U4 small nuclear RNPs, the U2AF(65), and serine/arginine-rich proteins. The splicing factors and the transcription machinery co-purify through a gel filtration column and co-immunoprecipitate in experiments using an anti-U2AF(65) antibody, indicating that they are part of a unique complex. Although the RNA Pol II-containing complex does not possess splicing activity, it can complement small nuclear RNP-inactivated extracts and can promote the formation of a pre-spliceosome complex. Because interactions between components of the splicing and transcription machineries occur in the context of a complex containing a hypophosphorylated RNA Pol II capable of initiating transcription, our results suggest that the coupling between transcription and splicing begins before transcription initiation.

Chromatography, Affinity↗

PubMed1997

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Amino Acid Sequence↗

PubMed1996

RNA-protein complexes.

The three commonly found RNA-binding domains, the ribonucleoprotein (RNP) domain, the double stranded RNA binding domain (dsRBD) and the K homology (KH) domain, have now been shown to have an alpha/beta fold similar to that found in many ribosomal proteins. Crystal structures of two hairpin RNA-protein complexes have been determined recently: the U1A spliceosomal protein bound to hairpin II of U1 small nuclear RNA, and MS2 bacteriophage capsid protein bound to a hairpin present at the ribosomal binding site of MS2 replicase mRNA. The crystal structure of the tryptophan operon RNA binding attenuation protein from Bacillus subtilis shows a novel structure with 11 monomers arranged in a doughnut-shaped ring that binds 11 copies of (U/G)AG triplets presented in the leader sequence of the tryptophan operon polycistronic message.

Animals↗

PubMed1991

Brain-specific small RNA transcript of the identifier sequences is present as a 10 S ribonucleoprotein particle.

BC-1 RNA is a small RNA transcript of the identifier repetitive sequences present in rodent genomes. The RNA has been reported to be specific to the brain and confined to the cytoplasm. The RNA level increases during the 1st month after birth. To understand its cytoplasmic function, it seems important to examine whether BC-1 RNA is present as an RNP. It is believed that the protein component may govern the functions of BC-1 RNA in the brain cells. In the present report, we have demonstrated that BC-1 RNA is not free but complexed with proteins to form a 10 S RNP in the cytoplasm. We have also shown that the 10 S RNP is not associated with cytoplasmic structures such as polysomes/ribosomes or microsomes. The buoyant density of the RNP was 1.26 g/cm3 in metrizamide. Furthermore, some of the protein components were shown to be in direct contact with RNA, since photo-cross-linking adducts of protein to BC-1 RNA were identified upon UV irradiation of the 10 S BC-1 RNP.

Animals↗