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PubMed1997

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

Animals↗

PubMed1987

Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.

Animals↗

PubMed1989

Ultrastructural distribution of ribonucleoprotein complexes during mitosis. snRNP antigens are contained in mitotic granule clusters.

The great majority of snRNP and hnRNP ribonucleoproteins have been shown to be confined to the nucleus except during periods of cell division. We have now determined the fine structure distribution of polypeptides associated with these RNP complexes during interphase and mitosis in mammalian tissue culture cells using immunoelectron microscopy. Many hnRNP antigens are found at the periphery of heterochromatin masses, known to be the sites of non-rRNP proteins initially surround areas of condensing chromatin and later become generally dispersed throughout the mitotic cell. The Sm protein antigens of snRNP complexes are found diffusely distributed in interphase nuclei as well as concentrated in fields of interchromatin granules (ICG). Proteins of snRNP complexes, unlike those of hnRNP, are associated with discernible cellular structures during mitosis. By prometaphase/metaphase, dense granular clusters are observed to contain a high concentration of snRNPs. These mitotic granule clusters (MGCs) are often in close proximity to chromosomal masses by late anaphase/telophase. The MGC structures are morphologically similar to interchromatin granule fields found in interphase nuclei. Furthermore, like interchromatin granules, they are sites of a high concentration of snRNP antigens and do not contain detectable hnRNP proteins or DNA.

Antigens↗

PubMed1979

Characterization of protein components of poly(A)-containing messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina.

Conditions for the isolation of intact poly(A)+mRNP from cryptobiotic gastruale of A. salina are described. In the presence of Mg2+ ions nucleolytic cleavage occurs in vitro in the vicinity of the 3'-poly(A) segment of mRNP during the isolation procedure. The resulting two parts of poly(A)+mRNP complex are separated by thermal elution from oligo(dT)-cellulsoe affinity column. Analysis by SDS-gel electrophoresis of protein components associated with intact poly(A)+mRNP has revealed the existence of 20--30 S RNP complex containing five major proteins with Mr 68,000, 53,000, 50,000, 45,000 and 38,000, respectively, but completely lacking the poly(A)-specific Mr 76,000 protein.

Animals↗

PubMed1987

Disassembly and domain structure of the proteins in the signal-recognition particle.

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum. SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2+-depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA. The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 micrograms/ml resulted in partial loss of activity, while 10 micrograms/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.

Animals↗

PubMed1990

The synthesis of influenza virus negative-strand RNA takes place in insoluble complexes present in the nuclear matrix fraction.

The replication of influenza virus RNA in vitro has been studied by cell fractionation of MDCK-infected cells and characterization of in vitro synthesized RNA. Analysis of the RNA product polarity by liquid hybridization to excess single-stranded DNA probes shows that only the RNP complexes present in the nuclear matrix fraction are able to synthesize negative-polarity RNA. This RNA product has been characterized as authentic vRNA by size analysis, RNase-protection by unlabelled, positive-polarity riboprobes and T1-fingerprinting. Priming the in vitro reaction with ApG stimulates preferentially the synthesis of positive-polarity RNA, while ApGpU stimulates both positive and negative-polarity RNA synthesis.

Animals↗

PubMed2006

Cryo-electron microscopy of spliceosomal components.

Splicing is an essential step of gene expression in which introns are removed from pre-mRNA to generate mature mRNA that can be translated by the ribosome. This reaction is catalyzed by a large and dynamic macromolecular RNP complex called the spliceosome. The spliceosome is formed by the stepwise integration of five snRNPs composed of U1, U2, U4, U5, and U6 snRNAs and more than 150 proteins binding sequentially to pre-mRNA. To study the structure of this particularly dynamic RNP machine that undergoes many changes in composition and conformation, single-particle cryo-electron microscopy (cryo-EM) is currently the method of choice. In this review, we present the results of these cryo-EM studies along with some new perspectives on structural and functional aspects of splicing, and we outline the perspectives and limitations of the cryo-EM technique in obtaining structural information about macromolecular complexes, such as the spliceosome, involved in splicing.

Cryoelectron Microscopy↗

PubMed1982

Isolation and characterization of hnRNA-snRNA-protein complexes from Morris hepatoma cells.

Of the RNA labelled after incubation of hepatoma cells with radioactive precursors for 20 and 150 min. 35% and 70%, respectively, can be isolated from nuclei by two consecutive extractions with 0.14 M NaCl at pH 8. The isolated RNA is complexed with nuclear proteins forming structures with sedimentation coefficients of less than 30 S to greater than 100 S. Similar complexes from rat liver isolated under the same experimental conditions show coefficients of 30-40 S. The RNA-associated proteins are similar, on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis, to the respective proteins of other cell types. The presence on these RNP complexes of six discrete small nuclear RNAs (snRNA) has been established. Experiments with a reversible inhibitor of RNA synthesis, D-galactosamine, demonstrated, differences in the turnover of hnRNA and snRNA. The half-lives of the six snRNA species has been determined, varying from 32 h for snRNA species a, b and d, to 22 h for snRNA species e and f and to 13 h for snRNA species c. Treatment of the nuclear extracts with 0.7 M and 1 M NaCl results in dissociation of hnRNA from the 'core' and other polypeptides, whereas snRNA remains complexed with polypeptides of Mr 54 000-59 000. Incubation of the nuclear extracts at 0 C with low doses of pancreatic R Nase (up to 1.5 micrograms/ml), which renders approximately 80% of the hnRNA acid-soluble and cleaves most of the snRNA, results in conversion of the high-molecular-weight hnRNPs to 30-S structures, without disrupting the 30-S RNP. Treatment of the nuclear extracts with higher doses of RNase (3 micrograms/ml) leads to disruption of the 30-S RNP and release of the hnRNA-associated proteins, underlining the importance of hnRNA-protein interaction for the retainment of the hnRNP structures.

Animals↗

PubMed1990

Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process.

To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.

Adenosine Triphosphate↗

PubMed1988

Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma).

Immunofluorescence on rat liver sections was used to select high-titer antinucleolar antibodies (ANoA) in the sera of patients with systemic sclerosis (scleroderma). In 646 patients, 53 ANoA sera (8%) were identified, and of these, 46 were available in sufficient quantities for further analysis. The complex of RNA polymerase I was immunoprecipitated by 7 sera (15%), which uniformly produced punctate nucleolar staining. The PM-Scl antigen, a particle consisting of 11 polypeptides, was immunoprecipitated by 8 sera (17%), all of which displayed homogeneous nucleolar staining. A 34-kd nucleolar protein (fibrillarin) of the U3 RNP complex was positive in immunoblotting of 22 sera (48%), which characteristically produced clumpy nucleolar staining. Antibodies against RNA polymerase I were associated with diffuse scleroderma of short duration, which was characterized by a high prevalence of internal organ involvement, including renal crisis. Anti-U3 RNP antibodies had a high prevalence in men with significantly less joint involvement, compared with ANoA-negative patients. Anti-PM-Scl antibodies identified a group of scleroderma patients with a high prevalence of concomitant myositis and renal involvement.

Antibodies, Antinuclear↗

PubMed1999

Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3' untranslated region determinant and poly(C) binding protein alphaCP.

Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human alpha-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3' untranslated region (3'UTR), the alpha-complex. The proteins of the alpha-complex are widely expressed. The alpha-complex or a closely related complex also assembles at pyrimidine-rich 3'UTR segments of other stable mRNAs. These data suggest that the alpha-complex may constitute a general determinant of mRNA stability. One or more alphaCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the alpha-complex. The ability of alphaCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the alpha-complex is a multisubunit structure. In the present study, we have addressed the composition of the alpha-complex. An RNA titration recruitment assay revealed that alphaCPs were quantitatively incorporated into the alpha-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major alphaCP isoforms and the alpha-globin 3'UTR was detected, suggesting that each of these proteins might be sufficient for alpha-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant alphaCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the alpha-complex could accommodate only a single alphaCP. These data established that a single alphaCP molecule binds directly to the alpha-globin 3'UTR, resulting in a simple binary structure for the alpha-complex.

3' Untranslated Regions↗

PubMed1992

Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins.

Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.

Amino Acid Sequence↗

PubMed1999

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.

Animals↗

PubMed2006

[The fragile X syndrome: one protein missing and 1001 disoriented mRNAs].

Fragile X syndrome is the most common form of inherited mental retardation. This X-linked disease is due to transcriptional silencing of the Fragile Mental Retardation 1 (FMR1) gene and the absence of its gene product, FMRP. This protein is an RNA-binding protein present in mRNP complexes associated with the translation machinery and is thought to be a key player in the control of mRNA transport in neurons. However, the exact role of FMRP in translation remains unclear. Two homologous proteins, FXR1P and FXR2P, are also found in RNP complexes containing FMRP, suggesting that FMRP's functions are much more complex than first thought. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated, as well as the putative roles of FXR1P and FXR2P as compensatory molecules. Here, we review the various possible functions of FMRP in RNA localization and transport in highly differentiated cells containing dendritic extensions such as neurons.

Fragile X Messenger Ribonucleoprotein 1↗

PubMed2004

Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity.

Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKepsilon kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKepsilon activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKepsilon following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKepsilon kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKepsilon in epithelial cells. Induction of IKKepsilon potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

Cell Line, Tumor↗

PubMed1996

Hierarchical self-tolerance to T cell determinants within the ubiquitous nuclear self-antigen La (SS-B) permits induction of systemic autoimmunity in normal mice.

Systemic autoimmune diseases are frequently associated with clustering of high titer autoantibody responses towards nuclear self-antigens. Little is known, however, about the extent of immune tolerance to the target nuclear antigens or the events leading to the complex autoantibody responses that are characteristic of systemic autoimmunity. To address these issues, we have examined the mouse immune response to La autoantigen (mLa) and the homologous human La antigen (hLa), which are components of the La(SS-B)/Ro(SS-A) ribonucleoprotein (RNP) complex targeted in systemic lupus erythematosus and primary Sjögren's syndrome. The findings reveal the presence of hierarchical T cell tolerance involving multiple autodeterminants within the La autoantigen expressed by normal H-2k and H-2a mice. At one end of this spectrum, there was no detectable T or B cell autoimmunity observed in mice that were immunized with the immunodominant mLa287-301 determinant, which differed by a single residue in its core sequence from the homologous but highly immunogenic human La288-302 determinant. Interestingly, the mLa287-301 peptide acted as an altered peptide ligand that specifically antagonized the activation of an hLa288-302-specific T cell hybridoma. In contrast to the tolerogenic mLa287-301 determinant, a range of autoimmune potential was identified among poorly tolerizing, subdominant self-peptides present within mouse La autoantigen. Notably, immunization of normal mice with the autologous subdominant La25-44 and La106-129 determinants resulted in limited or no detectable autoantibody response. In contrast, immunization with the subdominant mouse La13-30 determinant induced a proliferative T cell response associated with the appearance of specific autoantibodies recognizing multiple intrastructural (La) and intermolecular components (Ro) of the murine La/Ro RNP. The findings suggest how diversified autoimmunity might follow initiation of immunity to simple peptide mimics of poorly tolerogenic determinants that are present within ubiquitous self-antigens.

Amino Acid Sequence↗

PubMed2005

RNA chaperone activity of protein components of human Ro RNPs.

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.

Autoantigens↗

PubMed1994

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Base Sequence↗