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PubMed1996

Assembly of a ribonucleoprotein catalyst by tertiary structure capture.

CBP2 is an RNA tertiary structure binding protein required for efficient splicing of a yeast mitochondrial group I intron. CBP2 must wait for folding of the two RNA domains that make up the catalytic core before it can bind. In a subsequent step, association of the 5' domain of the RNA is stabilized by additional interactions with the protein. Thus, CBP2 functions primarily to capture otherwise transient RNA tertiary structures. This simple one-RNA, one-protein system has revealed how the kinetic pathway of RNA folding can direct the assembly of a specific ribonucleoprotein complex. There are parallels to steps in the formation of a much more complex ribonucleoprotein, the 30S ribosomal subunit.

Catalysis↗

PubMed1991

Interactions between viral coat protein and a specific binding region on turnip crinkle virus RNA.

The turnip crinkle virus coat protein binding sites in the ribonucleoprotein complex resulting from virion dissociation have been identified previously. In this study, RNA binding characteristics of viral coat protein to a region encompassing the protected RNA fragments Fa, Ff, and Fc (Fafc) have been investigated further using an RNA transcript (the Fafc fragment). These experiments have shown that coat protein requires no additional viral RNA elements to bind to this region. Such binding was shown to be specific for turnip crinkle virus coat protein using an ultra-violet light cross-linking assay. Gel mobility shift analyses demonstrated that the protein-RNA interactions produced two complexes: a homogeneous small ribonucleoprotein complex, and larger complexes which failed to migrate into gels. High salt and limiting protein concentrations favored the formation of the small ribonucleoprotein complex, whereas low salt and excess protein concentrations favored the larger complexes. RNA competition experiments demonstrated that small ribonucleoprotein complex formation coincided with specific RNA binding of the coat protein to the Fafc fragment. In addition, the coat protein possessed a poly(U)-binding site(s), which enabled it to interact with single-stranded RNA in a sequence non-specific manner to form large complexes. The results suggest that the coat protein contains both specific and non-specific RNA binding activities located at physically distinct sites. These results are consistent with the proposed assembly model for turnip crinkle virus.

Binding Sites↗

PubMed2001

RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex.

Nuclear export of ribonucleoprotein complexes requires cis-acting signals and recognition by receptors that mediate translocation through the nuclear pore complex. Translocation is likely to involve a series of physical interactions between the ribonucleoprotein complex and nucleoporins within the nuclear pore complex. Here, we have characterized the function of NXT1 in the context of the Tap-dependent RNA export pathway. Tap has been implicated in the nuclear export of RNA transcripts derived from Mason-Pfizer monkey virus that contain the constitutive transport element. We demonstrate that NXT1 stimulates binding of a Tap-RNA complex to nucleoporins in vitro, and we provide mutational analysis that shows these interactions are necessary for nuclear export of an intron-containing viral mRNA in vivo. Tap contains separate domains for binding to nucleoporins and NXT1, both of which are critical for its export function. RNA export is mediated by a heterodimer of Tap and NXT1, and the function of NXT1 on this pathway is to regulate the affinity of the Tap-RNA complex for nucleoporins within the nuclear pore complex. We propose that NXT1-dependent binding of the Tap-RNA complex to the nucleoporin p62, which we have reconstituted in vitro using recombinant proteins, represents a single step of the translocation reaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1981

Stage-specific synthesis of proteins complexed to ribonucleoprotein particles and ribosomes in zoospores of Blastocladiella emersonii.

In Blastocladiella emersonii zoospores, a set of proteins was found associated with the ribosomes and free ribonucleoprotein particles distinct from the ribosomes and polyribosomes. These proteins were designated P120, P105, P64, P56, and P42 based on their molecular weights determined by gel electrophoresis. Synthesis of these proteins was detected only during late sporulation just before the time polyadenylated ribonucleic acid accumulates in the sporangia. These proteins banded in isopycnic metrizamide gradients at densities of 1.31 and 1.27 g/cm3, which corresponded to the densities of the ribosomes and free ribonucleoprotein particles, respectively. Comparison of the distribution of the proteins in sucrose versus metrizamide gradients suggested that P105 was removed from the free ribonucleoprotein particles before complexing with the ribosomes. During germination, these proteins disappeared from the ribosomal fractions, with kinetics corresponding to the resumption of protein synthesis. Another protein (P178) was observed to bind to the ribosomes before the onset of protein synthesis during germination. Cycloheximide did not block the addition of this protein to the monoribosomes.

Blastocladiella↗

PubMed1998

cDNA cloning and characterization of the human U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein.

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.

Amino Acid Sequence↗

PubMed2002

Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins.

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.

Animals↗

PubMed1975

Electron microscopic observations on the poly(A)-RNP component of the pre-mRNA containing nuclear complexes.

The nuclear ribonucleoprotein particles containing polyadenylic acid were prepared from rat liver nuclear extract by treatment with RNase I. The sedimentation coefficient of the particles was approximately 14 S. These particles represent a component of the pre-mRNA containing ribonucleoprotein complexes of the nucleus. The submicroscopic morphology of the particles was examined in preparations pretreated by platinum-palladium shadowing and by various negative staining methods as well as in ultrathin sections. The 14 S particles are globular structures, 120-140 A in diameter. The particles are characterized by a strong tendency to aggregation. The results are discussed with respect to mRNA transport.

Animals↗

PubMed1997

Characterization of human telomerase complex.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called "telomeres" to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

Amino Acid Sequence↗

PubMed2003

Annexin A2 is a novel RNA-binding protein.

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.

Annexin A2↗

PubMed1990

Leader sequence distinguishes between translatable and encapsidated measles virus RNAs.

The 3'-terminal 55 nucleotides of the negative-strand measles virus RNA genome called the leader sequence is not transcribed into a detectable distinct RNA product. Most of the monocistronic N and bicistronic N-P RNAs lack the leader sequence. However, a subpopulation of the N and N-P RNAs and all of the antigenomes possess this leader. Here, we show that leader-containing subgenomic RNAs are functionally distinct from their leaderless counterparts. In measles virus-infected cells, leaderless monocistronic N and bicistronic N-P RNAs were associated with polysomes. By contrast, leader-containing N and N-P RNAs were found exclusively in nonpolysomal ribonucleoprotein complexes that were resistant to RNase and had a buoyant density of 1.30 g/ml, the same as that of antigenomic ribonucleoprotein complexes. Both antigenomic and subgenomic ribonucleoprotein complexes were specifically immunoprecipitated by antiserum against the N protein, and leaderless RNAs were not found in these complexes. These findings suggest that measles virus distinguishes RNAs destined for encapsidation or translation by the presence or absence of a leader sequence.

Animals↗

PubMed2001

The La antigen associates with the human telomerase ribonucleoprotein and influences telomere length in vivo.

La is an important component of ribonucleoprotein complexes and telomerase is a ribonucleoprotein that compensates for the shortening of the ends of linear DNA by adding telomeric repeats onto the ends of chromosomes by using an integral RNA as the template. We have identified a direct and specific interaction between La and the RNA component of human telomerase. Antibodies specific to La precipitate the human telomerase ribonucleoprotein complex derived from tumor cells, telomerase immortalized normal cells, and in vitro transformed cells. Overexpression of La in both experimentally immortalized human cells and prostate cancer cells results in gradual telomere shortening. Our results demonstrate that La can associate with telomerase and its expression level can influence telomere homeostasis in vivo.

Aging↗

PubMed2000

Persistent yeast single-stranded RNA viruses exist in vivo as genomic RNA.RNA polymerase complexes in 1:1 stoichiometry.

Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins. Both RNAs form ribonucleoprotein complexes with their cognate polymerases. Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles. Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses.

Blotting, Northern↗

PubMed2003

Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA.

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.

3' Untranslated Regions↗

PubMed2005

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein.

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.

Active Transport, Cell Nucleus↗

PubMed2001

The Yin Yang 1 transcription factor associates with ribonucleoprotein (mRNP) complexes in the cytoplasm of Xenopus oocytes.

Yin Yang 1 (YY1) is a multifunctional transcription factor that activates, represses, or initiates transcription of a diverse assortment of genes. Previous studies suggest a role for YY1 in cellular growth and differentiation, but its biological function during development of the vertebrate oocyte or embryo remains to be determined. We recently showed that YY1 is abundantly expressed throughout oogenesis and early embryonic stages of Xenopus, but it is sequestered in the cytoplasm and does not function directly in transcriptional regulation. In the present study we used a series of biochemical analyses to explore the potential function of YY1 in the oocyte cytoplasm. YY1 was isolated from oocyte lysates by oligo(dT)-cellulose chromatography, suggesting that it associates with maternally expressed mRNA in vivo. RNA mobility shift assays demonstrate that endogenous YY1 binds to labeled histone mRNA. Size exclusion chromatography of oocyte lysates revealed that YY1 exists in high molecular mass complexes in the range of 480 kDa. Destruction of endogenous RNA by RNase treatment of lysates, abolished the binding of YY1 to oligo(dT)-cellulose and resulted in redistribution from 480-kDa complexes to the monomeric form. Microinjection of RNase directly into the cytoplasm released YY1 from 480-kDa complexes and unmasked its DNA-binding activity, but did not promote translocation to the nucleus. These results provide evidence that YY1 is a component of ribonucleoprotein (mRNP) complexes in the Xenopus oocyte, indicating a novel function for YY1 in the storage or metabolism of maternal transcripts.

Active Transport, Cell Nucleus↗

PubMed1979

Rearrangements in the course of ribonuclease hydrolysis of pre-messenger ribonucleoproteins. A warning.

A study of the action of ribonuclease on 30--50-S monoparticles prepared from pre-messenger ribonucleoprotein (pre-mRNA . protein) was started in order to elucidate the structure of monoparticles. A ribonucleoprotein complex containing mostly 30000--38000-Mr proteins of pI 7--9 (alpha class) persisted under conditions where other proteins (23000--110000 Mr, pI 5--8.5, beta class) were relased. An unexpected increase of sedimentation coefficient accompanied the formation of the ribonucleoprotein complex. The extent of increase varied with the initial size of the monoparticles, reaching 45% for 30-S monoparticles. The ribonucleoprotein complexes designated here as 40--45-S alpha-ribonucleoproteins were more homogeneous in size than the original monoparticles. Electron microscopic examination showed that the sedimentation shift corresponded to an increase of the actual size of the particles, not to flattening or change of shape. Therefore, the 40--45-S alpha-ribonucleoprotein is not a pre-existing unit of pre-mRNA . protein but arises from specific rearrangements probably between small alpha ribonucleoproteins formed by fragmentation of monoparticles. In addition to the 40--45-S alpha-ribonucleoproteins, large protein aggregates corresponding to 15% of the monoparticle proteins were formed upon ribonuclease hydrolysis. Their major proteins were neutral, suggesting that the aggregates might be precipitates of proteins at pH close to the pI. Ribonuclease being a widespread cellular enzyme, partial rearrangements may occur during preparation and handling of pre-mRNA . protein. It is particularly crucial to remark that the 40--45-S alpha-ribonucleoprotein which does not pre-exist might be mistaken for a pre-mRNA . protein unit.

Centrifugation, Density Gradient↗

PubMed1994

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.

Amino Acid Sequence↗

PubMed2006

Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes.

The 3'-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855-10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by ARE(bcl-2) (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to ARE(bcl-2) RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein-ARE(bcl-2) RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of beta-globin-ARE(bcl-2) transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.

3' Untranslated Regions↗