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PubMed2003

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes.

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.

Chromosomal Proteins, Non-Histone↗
PubMed1987

Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.

Animals↗
PubMed2005

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

Bornaviridae↗
PubMed2025

Purification and Ultramicroscopic Observation of the Influenza A Virus Ribonucleoprotein Complex.

Influenza A virus (IAV) has an eight-segmented, single-stranded, negative-sense viral genomic RNA (vRNA). Each vRNA strand associates with nucleoproteins and an RNA-dependent RNA polymerase complex to form a viral ribonucleoprotein (vRNP) complex. IAV vRNPs adopt a flexible double-helical configuration that varies in length. Although the transcription and replication of vRNA take place in the context of vRNPs, the precise structural conformation of vRNPs during RNA synthesis remains partially elucidated. To unravel the intricate ultrastructure of the vRNP, it is necessary to purify it while preserving its native functionality. Herein, we introduce a comprehensive protocol for the purification of IAV vRNPs using glycerol gradient ultracentrifugation. Furthermore, we provide a method for the high-speed atomic force microscopy observation of vRNPs during viral RNA synthesis.

Ribonucleoproteins↗
PubMed1997

Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.

3C Viral Proteases↗
PubMed1994

Molecular composition of Ro small ribonucleoprotein complexes in human cells. Intracellular localization of the 60- and 52-kD proteins.

Ro small ribonucleoprotein complexes (RoRNPs) are thought to comprise several proteins, including the 60-kD Ro and the 52-kD Ro proteins, and several small RNAs, designated Y RNAs. Although RoRNPs are fairly ubiquitous in nature, their precise composition remains unknown, their function has been elusive, and their intracellular localization has been controversial. We have analyzed HeLa cell extracts by glycerol density gradient fractionation in order to determine the distribution of the individual protein and RNA components of RoRNPs. We found that 52-kD Ro was not detectable in an RNP complex with the 60-kD protein under a variety of conditions. Pretreatment of cell extracts with ribonuclease affected gradient migration of the 60-kD but not the 52-kD protein, suggesting that the latter is not complexed with RNA. The migration of the hY RNAs in these gradients closely followed that of 60-kD and not 52-kD Ro. Immunofluorescence analysis of two different cell lines with monospecific antibodies against 52- and 60-kD proteins strongly suggests that these two proteins are not present on overlapping sets of structures in vivo. We conclude that the 52-kD Ro protein is not a detectable component of the RoRNP complex under these conditions despite its reactivity with Ro autoimmune antisera.

Autoantigens↗
PubMed1995

Y3 is the most conserved small RNA component of Ro ribonucleoprotein complexes in vertebrate species.

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Animals↗
PubMed1976

Synthesis of delta crystallin from embryonic chick lens messenger ribonucleoprotein complex.

A single, major 21 S messenger ribonucleoprotein (mRNP complex) was isolated and purified by sucrose gradient centrifugation after EDTA treatment of high salt washed polysomes from 15 day embryonic chick lenses. A 17 S mRNA was released from the 21 S subunit of delta crystallin. Similar results were obtained with the 17 S mRNA released from the 21 S mRNP complex.

Animals↗
PubMed1993

Subcellular distribution of Ro ribonucleoprotein complexes and their constituents.

Ro ribonucleoprotein particles (Ro RNPs) are complexes of several proteins with a small RNA polymerase III-transcribed Ro RNA. Despite their relative abundance and evolutionary conservation no function has as yet been ascribed to these complexes. Also their subcellular distribution is still largely unknown as immunofluorescence studies concerning their localization have produced conflicting data. We have used cell enucleation to fractionate cells into cytoplasmic and nuclear fractions. Analysis of these fractions revealed an exclusively cytoplasmic localization for the Ro RNPs. The majority of the Ro RNAs are shown to be stably associated with all three known Ro RNP proteins. Although no Ro RNAs could be detected in the nuclear fraction, the Ro RNP-specific proteins were abundantly present. These nuclear non-Ro RNA-associated proteins are shown to be capable of binding Ro RNAs.

3T3 Cells↗
PubMed1977

Characterization of the protein moiety of messenger ribonucleoprotein complexes from duck reticulocytes by two-dimensional polyacrylamide gel electrophoresis.

The protein moiety of duck globin messenger ribonucleoprotein complexes isolated by oligo(dT)-cellulose chromatography or by sucrose gradient centrifugation was analysed by two-dimensional polyacrylamide gel electrophoresis under conditions where the separation in the first dimension occurs according to charge and in the second according to molecular weight. By comparing the pattern of protein from the mRNA - protein complex with that of ribosomal subunits we found that two acidic proteins with an identical molecular weight of about 49 000 and three basic proteins of about Mr 56 000, 64 000 and 73 000 were associated with the duck globin mRNA but were absent from either puromycin/high-salt-derived or 'run-off' ribosomal subunits. The comparison of the proteins from the complex with mRNA with those found in the 0.5 M KCl wash, commonly used as the source of initiation factors, showed also that only the 49 000-Mr protein from the complex could possibly be present in the 0.5 M KCl wash of polyribosomes; proteins with mobilities similar to the other three proteins complexed with mRNA were not detected in the salt wash of polyribosomes.

Animals↗
PubMed1975

Nuclear ribonucleoprotein complexes containing U1 and U2 RNA.

Nuclear ribonucleoprotein (RNP) complexes that contain the U1 and U2 RNA of chromatin of Novikoff hepatoma cells were extracted with 0.01 M Tris-HCl (pH 8.0) after the nuclei were initially washed with 0.075 M NaCl and 0.025 M EDTA (pH 8.0). These RNP complexes were purified by chromatography on Sepharose 6B columns and centrifugation on sucrose density gradients. The identity of the U1 and U2 RNA in these particles was established by their electrophoretic mobility in polyacrylamide gels and their T1 RNase fingerprints which were identical with those of authentic U1 and U2 RNA (R. Reddy et al. (1974), J. Biol. Chem.249, 6486-6494; H. Shibata et al. (1974), Mol. Cell. Biochem. 4, 3-19). The nuclear riboncleoproteins had a buoyant density of 1.47 g/ml in CsCl gradients. Two-dimensional polyacrylamide gel electrophoresis of their proteins showed these RNP complexes contain 10 polypeptide spots, of which two are phosphorylated in vivo.

Animals↗
PubMed1999

RNA-protein interactions in the human RNase MRP ribonucleoprotein complex.

The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5' end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.

Apoptosis Regulatory Proteins↗
PubMed1984

Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.

The C proteins (C1 and C2) are major constituents of the 40S subparticle of heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) (Beyer, A.L., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon, 1977, Cell, 11:127-138) and are two of the most prominent proteins that become cross-linked by ultraviolet light to heterogeneous nuclear RNA (hnRNA) in vivo. Studies are described here on the characterization of the C proteins in vertebrate cells using monoclonal and polyclonal antibodies. Monoclonal antibodies to genuine RNP proteins, including the C proteins, were obtained by immunizing mice with purified complexes of poly(A)+ hnRNA and poly(A)+ mRNA with their contacting proteins in vivo obtained by ultraviolet cross-linking the complexes in intact cells (Dreyfuss, G., Y.D. Choi, and S.A. Adam, 1984, Mol. Cell. Biol., 4:1104-1114). One of the monoclonal antibodies identified the C proteins in widely divergent species ranging from human to lizard. In all species examined, there were two C proteins in the molecular weight range of from 39,000 to 42,000 for C1, and from 40,000 to 45,000 for C2. The two C proteins were found to be highly related to each other; they were recognized by the same monoclonal antibodies and antibodies raised against purified C1 reacted also with C2. In avian, rodent, and human cells the C proteins were phosphorylated and were in contact with hnRNA in vivo. Immunofluorescence microscopy demonstrated that the C proteins are segregated to the nucleus. Within the nucleus the C proteins were not found in nucleoli and were not associated with chromatin as seen in cells in prophase. These findings demonstrate that C proteins with similar characteristics to those in humans are ubiquitous components of hnRNPs in vertebrates.

Animals↗
PubMed1999

Formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein A1 and transcription-regulatory elements of viral RNA.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) specifically binds to two transcription-regulatory elements, i.e., the leader and intergenic sequence, of the negative-strand (template-strand) RNA of mouse hepatitis virus (MHV) and may play a role in viral RNA transcription. Previous studies based on the defective-interfering RNAs of MHV suggested that these two RNA elements may interact with each other during transcription, although they do not have complementary sequences. In this study, we showed by an in vitro reconstitution assay that hnRNP A1 could mediate the formation of an RNP complex involving these two RNA elements. Both the RNA-binding domains and protein-interacting domain of hnRNP A1 contributed to the efficient formation of the RNP complex; however, the presence of the two RNA-binding domains alone, without the protein-interacting domain, also resulted in some RNP formation. Omission of hnRNP A1 in the reconstitution reaction abolished the RNP formation, and mutations of the IG sequences significantly inhibited the RNP formation. These findings suggest that the two cis-acting transcription-regulatory sequences of MHV RNA can interact with each other through the formation of an RNP complex involving a cellular protein hnRNP A1. This RNP complex may participate in MHV RNA transcription.

Animals↗
PubMed1974

Nuclear ribonucleoprotein complexes containing polyadenylate from mouse ascites cells.

Nuclear poly(A)-containing RNA of mouse ascites cells can be extracted in the form of 15-17S ribonucleoprotein complexes under conditions in which the bulk of the heterogeneous nuclear RNA is released as 30S complexes. The poly(A)-containing fraction of nuclear extracts has been resolved into two distinct components, 15 and 17 S; neither contains the two polypeptides of 30S ribonucleoprotein. The 17S particle contains approximately six polypeptide species of molecular masses 17,000-30,000 daltons. The 15S complex has four distinct polypeptides of higher molecular mass, including a prominent 80,000-dalton species.

Adenine Nucleotides↗
PubMed1988

Monoclonal autoantibody recognizing a unique set of small nuclear ribonucleoprotein complexes.

A murine IgG2a, kappa-monoclonal autoantibody (mAb) F78 is described that recognizes a novel epitope associated with small nuclear ribonucleoprotein complexes (snRNP). F78 selectively immunoprecipitated a unique pattern of small nuclear RNA (U1, U2, and U4 to U6) characterized by a marked depletion of U1 and an elevated proportion of U2 compared with known patterns immunoprecipitated by previously described anti-RNP (2.73) and anti-Sm (7.13, Y12) mAb. Analysis of immunoprecipitated RNA from extracts previously cleared with mAb F78 and probed with anti-RNP mAb 2.73 further indicated the presence of two distinct subsets of U1. Immunoblots of whole cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without heating showed that F78 selectively bound to a trypsin-sensitive component of apparent m.w. greater than 120,000 which was decreased in size following RNase A treatment. The anti-Sm mAb, but not the anti-RNP mAb, also recognized this component in unheated samples. Heating before SDS-PAGE resulted in abrogation of binding to the F78 epitope. Immunoprecipitation of unlabeled or [35S]methionine-labeled cell extracts with F78 revealed the presence of most snRNP peptides, but the absence of peptide C and the 68,000 m.w. component, known to be selectively associated with U1-specific snRNP. Two-dimensional SDS-PAGE analysis of F78 immunoprecipitates confirmed that the epitope recognized by this mAb resides on a heat-dissociable complex containing snRNP-related peptides B, B', D, E, F, and G, but lacking U1-associated peptides. F78 mAb therefore defines a subset of snRNP which lack anti-RNP associated U1 RNA as well as peptides known to be selectively associated with this RNA species. It apparently recognizes an epitope associated with an assembled form of these particles and may be useful in examining structures involved in RNA processing.

Antibodies, Monoclonal↗
PubMed2005

A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly.

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

Animals↗
PubMed2005

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

Pseudouridine (Psi) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Psi synthase bind directly the sRNA; aCBF5 also interacts directly and independently with aNOP10 and aGAR1. Presence of aCBF5, aNOP10 and a U residue at the pseudouridylation site in the target RNA are required for RNA target recruitment. In agreement, we found that the aCBF5-aNOP10 pair is the minimal set of proteins needed for the formation of a particle active for pseudouridylation. However, particles more efficient in targeted pseudouridylation can be formed with the addition of proteins L7Ae and/or aGAR1. Although necessary for optimal activity, the conserved ACA motif in the sRNA was found to be not essential.

Archaeal Proteins↗