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PubMed2003

Interaction of cellular tubulin with Sendai virus M protein regulates transcription of viral genome.

Cellular tubulin has been shown to activate in vitro transcription with Sendai virus (SeV) particles. In this study, the molecular basis for the transcriptional activation by tubulin was investigated. We showed that tubulin dissociates viral matrix (M) protein, which acts as a negative regulator for transcription, from viral ribonucleoprotein (RNP) consisting of L, P, N proteins, and the genome RNA. Both alpha and beta subunits of human tubulin, which were expressed as GST fusion proteins, were found to stimulate viral mRNA synthesis similar to native alpha/beta-heterodimer tubulin. Pull-down assay using GST-tubulin subunits demonstrated that M protein is released from the RNP as a complex with each tubulin subunit. In vitro-binding analyses revealed that M protein directly interacts with tubulin as well as microtubules. These findings suggest that interaction of M protein with tubulin may have an important role in the regulation of SeV transcription.

Binding Sites↗

PubMed1998

GalR-mediated repression and activation of hybrid lacUV5 promoter: differential contacts with RNA polymerase.

The GalR repressor regulates expression of genes of the gal regulon in Escherichia coli. We studied the regulatory effect of GalR in vitro on a heterologous promoter, lacUV5, by placing the GalR-binding site, OE, at different locations upstream of this promoter. Despite the fact that the lacUV5 promoter is transcribed efficiently by RNA polymerase (RNP) alone, GalR modulated transcription from many of the PlacUV5 variants. Depending on the location of OE and the neighboring DNA sequence, GalR repressed, activated or had no effect on the promoter. Both repression and activation involved formation of GalR-RNP-DNA ternary complexes and required an intact c-domain of the alpha subunit of the holoenzyme. These results support the differential contact model of a regulator action, in which a regulator differentially binds to, and lowers the energy of, intermediates of transcription initiation either to hinder or to facilitate a step of initiation. The nature of the contacts depends upon the context, i.e. the geometry of the ternary complex. The observed repression and activation effect of GalR on a heterologous promoter also underscores the point that a regulator is not a dedicated protein for repression or for activation.

Binding Sites↗

PubMed1997

Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Antigens, Nuclear↗

PubMed2006

Probing the structure and function of an archaeal C/D-box methylation guide sRNA.

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.

Archaeal Proteins↗

PubMed2002

Association of influenza virus matrix protein with ribonucleoproteins may control viral growth and morphology.

The matrix protein (M1) of influenza virus plays a central role in viral replication. In relation to viral growth and morphology, we studied the RNP-binding activity of M1s from high-growth strain A/Puerto Rico/8/34 (A/PR8/34) and the relatively low-growth wild-type strain A/Nanchang/933/95. The RNP-binding strength of M1 was studied by disruption of M1 from M1/RNP complexes with salt and acidic condition. Our results indicated that binding of M1 of high-growth A/PR8/34 was more difficult to break than the binding of M1 of low-growth A/Nanchang/933/95. Consistent with the presence of M1 in A/PR8/34, binding of M1 of Resvir-9, a reassortant containing P, M, and NS genes from A/PR8/34 and the rest of genes from A/Nanchang/933/95 and retaining relative high-growth characteristic, was relatively difficult to break than the binding of M1 of A/Nanchang/933/95. Physical properties of morphological features of these viruses were studied by velocity sucrose gradient centrifugation and transmission electron microscopy of purified viral particles, and by immunofluorescence staining of hemagglutinin expressed on the surface of infected cells. The results demonstrated that high-growth strains, A/PR8/34, and a relative high-growth reassortant, Resvir-9, had characteristics associated predominantly with spherical particles, while the low-growth strain, A/Nanchang/933/95, had characteristics of filamentous particles. These studies indicate that the binding between M1 and RNP complex might determine viral growth and morphology.

Animals↗

PubMed2004

Lsm proteins promote regeneration of pre-mRNA splicing activity.

Lsm proteins are ubiquitous, multifunctional proteins that affect the processing of most RNAs in eukaryotic cells, but their function is unknown. A complex of seven Lsm proteins, Lsm2-8, associates with the U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs. Spliceosomes contain five snRNAs, U1, U2, U4, U5, and U6, that are packaged as ribonucleoprotein particles (snRNPs). U4 and U6 snRNAs contain extensive sequence complementarity and interact to form U4/U6 di-snRNPs. U4/U6 di-snRNPs associate with U5 snRNPs to form U4/U6.U5 tri-snRNPs prior to spliceosome assembly. Within spliceosomes, disruption of base-paired U4/U6 heterodimer allows U6 snRNA to form part of the catalytic center. Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, although little is known about this process. Here we present evidence that regeneration of splicing activity in vitro is dependent on Lsm proteins. RNP reconstitution experiments with exogenous U6 RNA show that Lsm proteins promote the formation of U6-containing complexes and suggest that Lsm proteins have a chaperone-like function, supporting the assembly or remodeling of RNP complexes involved in splicing. Such a function could explain the involvement of Lsm proteins in a wide variety of RNA processing pathways.

Multigene Family↗

PubMed1983

Nuclear bodies in mouse splenic lymphocytes: II - Cytochemistry and autoradiography during stimulation by concanavalin A.

Nuclear bodies (NB) are poorly understood nucleoplasmic structures frequently observed in many animal cell types. Murine lymphocytes mitogenically stimulated with concanavalin A contain 5 types of NB. In order to examine the origin and function of the NB we have carried out cell fractionation and have performed cytochemistry, immunocytochemistry and autoradiography at the electron microscope level. Regressive staining for RNP showed that simple NB (types I and II) and the shells of complex NB (types III, IVa and V) consist of a non-chromatinic fibrillar material which is most likely proteinaceous. Projections of this material from the NB surface appear to link the NB to nucleoplasmic fibrogranular elements. The fibrillar, filamentous and granular components of the cores of complex NB were largely RNP and in some instances closely resembled fibrogranular areas in the nucleoplasm. Chromatin masses were seen occasionally in the cores. All NB types remained unstained after nucleolus-specific silver nitrate staining, and were also not stained by a centromere-specific antibody. Autoradiography was performed along long-term labelling with 3H-uridine, 3H-thymidine or 3H-leucine. The NB were not labelled by any of the precursors, indicating that macromolecular synthesis does not play a major role in NB evolution in these cells. By fractionation, we showed that NB co-isolated with the nuclear matrix, and were linked to the fibrogranular nuclear matrix component by projections from the surface of the NB. A possible role of NB in RNA processing is discussed.

Animals↗

PubMed1995

An in vitro system for studying RNA-protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA.

Previous attempts to study the binding of yeast ribosomal protein L1 with 5S rRNA in vitro have been impeded by the failure to form RNA-protein complexes with purified protein and RNA. To circumvent this difficulty, we have developed an in vitro system that allowed RNP formation. The system involved in vitro expression of the protein L1 from its cloned gene in the presence of exogenous yeast 5S rRNA. A protein of the expected size (34 kDa) was synthesized by in vitro transcription and translation. A specific 5S rRNA-protein L1 complex (RNP) was formed when the rRNA molecule was present during protein L1 synthesis. However, the full-length protein L1 failed to bind 5S rRNA. The extent of RNP formation was proportional to the concentration of the exogenous yeast 5S rRNA in the reaction. The RNP displayed properties identical to those isolated from mature 60S ribosome subunits. Addition of yeast 5.8S rRNA did not result in the formation of a specific RNP. Using this in vitro system, we examined the ability of several deletion mutant proteins to bind yeast 5S rRNA and concluded that protein L1 missing residues 261 to 295 from the C-terminus could not bind yeast 5S rRNA. This in vitro system should be useful for future studies on the molecular nature of 5S rRNA-protein L1 interaction.

Protein Binding↗

PubMed1985

A multicomponent complex is involved in the splicing of messenger RNA precursors.

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.

Adenosine Triphosphate↗

PubMed1986

Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes.

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) within spread active chromatin has been investigated using specific anti-RNP antibodies. Monoclonal antibodies directed against the core proteins of heterogeneous nuclear (hn)RNP or against small nuclear (sn)RNP have been incubated directly with lysed mouse or Drosophila tissue culture cells and the bound antibodies visualized by means of a protein A-colloidal gold complex. The hnRNP core proteins have been localized on growing RNP fibrils within non-nucleolar transcription complexes. Anti-snRNP antibodies, directed either against the Sm-antigen (common for nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs) or against U1-snRNP, were bound by two morphological types of RNP structures. Within areas of chromatin that do not completely disperse, labeling was observed on RNP-fibril gradient type structures or on groups of fibrogranular material. In the well dispersed regions containing individual nonribosomal transcription complexes, snRNP antigens were associated with growing RNP fibrils. Our results provide direct evidence for association of some U-snRNP species (including U1-snRNP) with extranucleolar RNA as early as during transcription elongation. In addition, the presence of core hnRNP proteins on the same type of nascent RNA transcripts has been confirmed.

Animals↗

PubMed1993

Rare scleroderma autoantibodies to the U11 small nuclear ribonucleoprotein and to the trimethylguanosine cap of U small nuclear RNAs.

We have identified a scleroderma serum (Ru) with a previously undescribed specificity to protein components of the U11 small nuclear ribonucleoprotein particle (snRNP), a low-abundance member of the Sm class of U RNPs. The U11 RNP can be specifically immunoprecipitated from sonicated HeLa cells with Ru serum. In nuclear extracts, a fraction of the U11 particle is found complexed to the U12 RNP, an even lower abundance Sm snRNP. In glycerol gradient fractions, Ru serum identifies a 65-kDa protein that cosediments with the U11-U12 complex and is shifted upon targeted degradation of the U12 RNA. The 65-kDa protein therefore appears to be a component of the U11-U12 snRNP complex, whereas another Ru-reactive (140 kDa) protein may be associated with the free U11 RNP. The Ru serum also contains autoantibodies directed against the trimethylguanosine cap of U RNAs. This rare specificity has been described previously in only three other scleroderma patients.

Adult↗

PubMed2002

The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts.

Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors. Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif. Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs. Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e. Clf1Delta2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins. In vitro, temperature inactivation of Clf1Delta2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.

Base Sequence↗

PubMed1991

Two zinc finger proteins from Xenopus laevis bind the same region of 5S RNA but with different nuclease protection patterns.

Immature oocytes from Xenopus laevis contain a 42S ribonucleoprotein particle (RNP) containing 5S RNA, tRNA, a 43 kDa protein, and a 48 kDa protein. A particle containing 5S RNA and the 43 kDa protein (p43-5S) liberated from the 42S particle upon brief treatment with urea can be purified by anion exchange chromatography. The purified p43-5S RNA migrates as a distinct species during electrophoresis on native polyacrylamide gels. Radiolabeled 5S RNA can be incorporated into the p43-5S complex by an RNA exchange reaction. The resulting complexes containing labeled 5S RNA have a mobility on polyacrylamide gels identical to that of purified p43-5S RNPs. RNP complexes containing 5S RNA labeled at either the 5' or 3' end were probed with a variety of nucleases in order to identify residues protected by p43. Nuclease protection assays performed with alpha-sarcin indicate that p43 binds primarily helices I, II, IV, and V of 5S RNA. This is the same general binding site observed for TFIIIA on 5S RNA. Direct comparison of the binding sites of p43 and TFIIIA with T1 and cobra venom nucleases reveals striking differences in the protection patterns of these two proteins.

Animals↗

PubMed1998

Influenza virus nucleoprotein interacts with influenza virus polymerase proteins.

Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the COOH terminus of NP. Analysis of NP deletion mutants revealed that at least three regions of NP interacted independently with PB2. A detailed analysis of the COOH terminus of NP by mutation of serine-to-alanine (SA) residues either individually or together demonstrated that SA mutations in this region did not affect the binding of NP to PB2. However, some SA mutations at the COOH terminus drastically affected the functional activity of NP in an in vivo transcription-replication assay, whereas others exhibited a temperature-sensitive phenotype and still others had no effect on the transcription and replication of the viral RNA. These results suggest that a direct interaction of NP with polymerase proteins may be involved in regulating the switch of viral RNA synthesis from transcription to replication.

Nucleocapsid Proteins↗

PubMed2002

Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly.

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74 kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74 kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5' exon-intron part, rather than the intron-3' exon part, of pre-mRNA was observed.

Electrophoresis, Polyacrylamide Gel↗

PubMed1986

Differential chromosomal distribution of ribonucleoprotein antigens in nuclei of Drosophila spermatocytes.

The ribonucleoprotein (RNP) composition of the active Y chromosomal structures in spermatocyte nuclei of Drosophila hydei has been investigated using the anti-RNP antibodies Dm 28K2 and pp60 as a probe. Antibody Dm 28K2 was raised against an RNP protein of cytoplasmic RNP particles in D. melanogaster cells, while antibody pp60 was raised against a pre-messenger RNP fraction from oocytes of Xenopus laevis. Both antibodies detect nuclear RNP (nRNP) antigens of D. hydei. This is shown by CsCl density centrifugation of nRNP from D. hydei cells and immunoblotting across the density gradient. Dm 28K2 and pp60 recognize antigens of nRNP complexes which band at a characteristic buoyant density of approximately 1.4 g/cm3 in CsCl. By indirect immunofluorescence we observe that the nRNP complexes identified by Dm 28K2 are localized at only two of the five Y chromosomal loop structures which are named according to their distinct morphology. Dm 28K2 decorates RNPs within the "clubs," within the cones, and within the matrix of the "pseudonucleolus." Ultrastructural bodies that are candidates for this immunoreaction are RNP granules that resemble the so-called perichromatin granules. Antibody pp60 recognizes RNP complexes close to the axes of the active Y chromatin. In the "pseudonucleolus" it can be shown that the structures recognized by pp60 are quite distinct from those detected by Dm 28K2. Thus, the "pseudonucleolus" is a striking example for the presence of different RNP populations within a same defined nuclear compartment. Together with previous results (Glätzer, K. H., 1984, Mol. Gen. Genet., 196:236-243), our data represent evidence that the morphological and apparently functional differences between the active Y chromosomal loops, which are involved in male fertility, are caused by the presence of qualitatively and possibly also functionally different RNP populations within these nuclear compartments. Because both RNP antigens are discussed in the literature in connection with repressed mRNP the observed cross-reaction of the respective antibodies in D. hydei suggests a more general and important function of these proteins in the RNA metabolism of eukaryotic cells.

Animals↗

PubMed1984

The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific ScRNA and a characteristic set of proteins.

A novel ribonucleoprotein (RNP) particle showing a highly compact and characteristic structure in the electron microscope was found associated with globin and other repressed mRNA in the cytoplasm of duck, mouse and HeLa cells. This 19S complex is of extraordinary stability: dissociated by 0.5 M KCl or EDTA from the (still repressed) core globin mRNP, it can be purified on gradients containing 1% Sarkosyl, and resists (unfixed) caesium sulphate-dimethylsulphoxide density centrifugation. Its density of 1.31 g/cm3 indicates an RNP complex with a 15% RNA component. In mouse and duck it contains approximately 10 proteins in the 20 000-30 000 mol. wt. range, a few components of 50 000-70 000 mol. wt., and two specific small cytoplasmic RNAs (ScRNA) of 70-90 nucleotides. Both of these RNAs have identical 3'-terminal oligonucleotides. We propose the name 'prosome' for this ScRNP particle which somehow participates in negative control of mRNA translation, and we believe will prove to be ubiquitous to animal species.

Animals↗

PubMed1977

Properties of a nuclear polyadenylate-protein complex from mouse ascites cells.

Ribonucleoprotein (RNP) subcomplexes containing at least 60% of the total nuclear poly(A) were isolated from mouse ascites cells; these 15 S ribonucleoprotein particles were most probably derived from larger hnRNA containing complexes. The buoyant density of the 15 S ribonucleoprotein in formaldehyde-CsCl was significantly less than that of the hnRNA-containing 30 S ribonucleoprotein subcomplex, indicating a higher proportion of protein (approximately 80%) in the 15 S ribonucleoprotein. The proteins partially protected the poly(A) from the action of ribonuclease T2. RNA isolated from purified 15 S ribonucleoprotein was estimated to be 190 to 200 nucleotides in length by gel electrophoresis, whereas the RNA from crude preparations was slightly larger. Base composition analysis of 32P-labeled crude 15 S RNP-RNA showed it to be rich in adenylate (70%) but containing a substantial amount of uridylate (20%). The base composition of RNA from purified complexes was approximately 90% adenyalte. Our results suggested that oligo(U) sequences from hnRNA could artificially associated with the poly(A) during preparation of 15 S RNP-RNA.

Animals↗