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PubMed2002

Shared protein components of SINE RNPs.

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA-protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.

Base Sequence↗

PubMed1997

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs.

Bacterial Proteins↗

PubMed1978

[Nuclear and cytoplasmic ribonucleoprotein complexes of normal mouse liver cells, Guelstein hepatomas 22A and 48 and of liver of tumor-bearing animals].

Nuclear and cytoplasmic RNP complexes obtained from normal mouse liver cells, Guelstein hepatomas of different degrees of malignancy (22A and 48) as well as from liver of tumor-bearing mice were subjected to chromatography on a celite column (NPC--chromatography). In addition cytoplasmic RNP complexes were centrifuged in sucrose and CsCl density gradients. The results of the NPC-chromatography indicate that nuclear rapidly labelled RNA species of all tissues under study are constituents of the two main types of RNP particles differing from each other by the tightness of RNA-protein bonds. No precursor-product relationship could be revealed between the above types of RNP-particles of nucleus, labelled under conditions of a partial Actinomycin D block. Rapidly labelled nonribosomal cytoplasmic RNAs represent constituents of RNP-particles resembling nuclear ones in their degree of heterogeneity and chromatographic position. Sedimentation analysis of cytoplasmic RNP-particles from tumours showed an increase in relative proportion of monoribosomes and informosomes (free non-ribosomal cytosol RNP-complexes) at the expense of polyribosomes and mRNP complexes. Thus, the liver cells of experimental tumour-bearing animals undergo changes (although not very well-defined), typical for tumour cells.

Animals↗

PubMed1986

Solid-phase processing of U2 snRNA precursors.

HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.

Antibodies↗

PubMed1994

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

Animals↗

PubMed1994

[The epidermal growth factor induces specific changes in the expression of small RNA and of the set of small RNP in A-431 cells].

Specific small ribonucleoprotein (alpha-RNP) complexes have been identified and characterized in the human epidermal carcinoma A-431 cells. The alpha-RNP complexes contain Alu-homologous small RNA, along with other small antisense RNA species. The epidermal growth factor (EGF) has been shown to induce selective specific changes in the expression of the small alpha-RNAs, the expression of the Alu-like RNA being repressed. Specific changes in the protein composition of the alpha-RNP complexes have been detected under the influence of EGF.

Carcinoma, Squamous Cell↗

PubMed1995

Two immunologically related polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear RNP.

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.

Animals↗

PubMed1991

A novel 40S multi-snRNP complex isolated from rat liver nuclei.

Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.

Animals↗

PubMed1993

HnRNP CBP35.CBP67 interaction during stress response and ageing.

Previous studies have demonstrated the existence of nuclear carbohydrate binding proteins in a variety of mammalian cells with molecular masses of 35,000, 67,000, and 70,000 (CBP35, CBP67, and CBP70), which are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a carboxyl-terminal portion homologous to beta-galactoside-specific lectins. CBP35 it has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here we show that the exposure of mature rats to stress induces an increase in nuclear CBP35 bound to CBP67 and retained on immobilized glucose. Nuclear extracts from the livers of old rats displayed no detectable stress response. This CBP35.CBP67 association detected in rat liver is considered with respect to the CBP35.CBP70 association recently observed in HL60 cell nuclear extracts.

Aging↗

PubMedDate not supplied

[CBP35-CBP67 interaction in stress response and aging].

Three carbohydrate-binding proteins with relative molecular masses of 35, 67, and 70 kDa (CBP35, CBP67, and CBP70) have been described to be present in nuclei of mammalian cells, where they are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an N-terminal domain that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a C-terminal domain that is homologous to beta-galactoside-specific lectins. CBP35 has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here, we show that exposition of mature rats (6-8 months old) to stress results in binding of nuclear CBP35 to CBP67 which is retained on a column containing immobilized glucose. In contrast to mature animals, nuclear extracts from the livers of old rats (22-24 months old) displayed no detectable stress response.

Aging↗

PubMed2006

Protein-protein and protein-RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs.

The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.

Amino Acid Sequence↗

PubMed1998

Generation of influenza transfectants using purified recombinant nucleocapsid protein.

Affinity-purified type A influenza virus nucleocapsid protein expressed by a recombinant baculovirus vector was used in in vitro RNA transcription reactions to create RNP complexes containing a synthetic influenza A virus NS gene. When used in transfection assays, the baculovirus-expressed NP was shown to be biologically active allowing the efficient isolation of transfectant viruses containing the artificially-introduced NS gene. The results demonstrate that NP is the only virion protein necessary in the reconstituted RNP complexes used for transfection thus eliminating the need for purified RNP complexes containing active polymerase.

Baculoviridae↗

PubMed1985

Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates.

Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.

HeLa Cells↗

PubMed1991

Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis.

To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS1. We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication. Because NS1 and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis. We showed that virion RNP complexes phosphorylated NS1 but possessed no autophosphorylating activity. Soluble NP protein derived from RNP complexes did not phosphorylate NS1, but did phosphorylate casein. When NP protein was dephosphorylated, however, it no longer phosphorylated casein. We also showed that NS1 was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation.

Animals↗

PubMed1997

The selection in vivo and characterization of an RNA recognition motif for spectinomycin.

Ribonucleoprotein (RNP) complexes participate in almost all macromolecular processes, including RNA processing, protein synthesis, and the signal recognition of proteins targeted for export. An understanding of these processes requires detailed knowledge of interactions at the molecular level, which has evidently been difficult due to the size and complexity of the particles. Fragmentation of large RNP complexes into functional subdomains is proven to be a successful in vitro strategy to probe ligand interactions at the molecular level. We reasoned that RNA molecules expressed in vivo may fold in such a manner as to mimic a drug binding site present on the intact ribosome. If expressed at sufficient levels, the RNA would sequester the antibiotic thereby permitting the continued function of the ribosome and consequently allow the cell to survive in the presence of the drug. Evidence is presented here in support of this RNA fragment-rescue concept following the selection and characterization of RNA fragments that confer resistance to the antibiotic spectinomycin.

Anti-Bacterial Agents↗

PubMed1998

RNA-induced changes in the activity of the endonuclease encoded by the R2 retrotransposable element.

R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods. The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3' hydroxyl group generated from this nick to prime reverse transcription of its own RNA. This reaction has been termed target-primed reverse transcription (TPRT). Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA. In this study, more sensitive in vitro assays have enabled further characterization of these reactions. R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths. Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex. While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA. Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl. Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site. The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.

Animals↗

PubMed2001

Poliovirus RNA replication requires genome circularization through a protein-protein bridge.

The mechanisms and factors involved in the replication of positive stranded RNA viruses are still unclear. Using poliovirus as a model, we show that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the viral genome is necessary for RNA replication. Initiation of negative strand RNA synthesis requires a 3' poly(A) tail. Strikingly, it also requires a cloverleaf-like RNA structure located at the other end of the genome. An RNP complex formed around the 5' cloverleaf RNA structure interacts with the poly(A) binding protein bound to the 3' poly(A) tail, thus linking the ends of the viral RNA and effectively circularizing it. Formation of this circular RNP complex is required for initiation of negative strand RNA synthesis. RNA circularization may be a general replication mechanism for positive stranded RNA viruses.

Base Sequence↗

PubMed1975

Induced RNP production in different cell types of Drosophila.

The typical RNP complexes produced in puff 2-48BC in the salivary gland chromosomes as a consequence of treatments interfering with the cellular respiratory metabolism have been found in various other cell types after treatment with 5-10- minus 2 M vitamin B6 for 4 hr. In Drosophila hydei the typical RNP complexes were present in puff 2-48BC in the nuclei of gastric caeca cells, in nuclei of cells of the imaginal (wing) discs and embryonic cells in primary culture. Similar RNP complexes were observed in a puff in D. virilis salivary gland nuclei, but not in salivary gland nuclei of D. melanogaster.

Animals↗