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Large nuclear RNP particles--the nuclear pre-mRNA processing machine.

Processing of nuclear pre-mRNA is an important step in the regulation of gene expression and involves 5(')- and 3(')-end processing, splicing, and editing. Mammalian nuclear pre-mRNAs are assembled in large ribonucleoprotein (lnRNP) complexes, in which the entire population of nuclear pre-mRNA is individually packaged until it is exported to the cytoplasm. The lnRNP particles are supraspliceosomal complexes. They are composed of four spliceosomal substructures and an additional one, which are interconnected by the pre-mRNA, and have an overall mass of 21MDa. The additional substructure was proposed to harbor additional processing activities, such as editing components that were shown to be associated with the lnRNP particles. Here we show that the cap-binding proteins (CBPs), CBP20 and CBP80, are associated with the lnRNP particles, as well as components of the 3(')-end-processing activity. These results, together with our previous demonstration of the association of splicing factors and A-to-I editing enzymes with lnRNP particles, support the view that the lnRNP particles are the nuclear pre-mRNA processing machine. Such a machine is required to execute the nuclear processing steps of the pre-mRNA in an accurate and regulated manner. The supraspliceosomal pre-mRNA processing machine, in which each substructure represents a functional spliceosome, provides a frame onto which the pre-mRNA is folded. It allows juxtaposition of exons about to be spliced, while introns are looped out of each of the respective spliceosomes. This model can account for regulated alternative splicing, which is a major source of protein versatility in mammals.

Blotting, Western↗

PubMed1981

Organization of the nucleolar RNP components in cultured rabbit fibroblasts following mild loosening of the nuclear content.

The organization of the ribonucleoprotein (RNP) components of the nucleolus was studied in ultrathin sections by a recent method of mild loosening of the nuclear content. This investigation was carried out in cultured rabbit fibroblasts in which the nucleoli contain clearly separated fibrillar and granular RNP components. In addition, the demarcation between these two particulate components was accentuated by infection with herpes simplex type 1 virus (HSV1), by recovery at 37 degrees C after a heat shock, and by actinomycin D treatment. Except in actinomycin D-treated cells, it was possible to identify two distinct components which are not visible in routinely prepared sections: (1) highly contrasted fibrillar clusters resulting from the loosening of the fibrillar component, (2) a granular network composed of nucleolar granules interconnected by a thin filament of about 5 nm in thickness. In actinomycin D-treated cells, only the granular zone was observed. High resolution autoradiography carried out both on standard fixed and on loosened cells following a 5 min labeling with tritiated uridine revealed that the fibrillar clusters correspond to sections of nucleolar transcription complexes. When the same labeling was followed by 3 h chase only the granular network was labeled indicating that it corresponds to the site of pre-ribosomal ribonucleic acid (pre-rRNA) maturation. Infection by HSV1 did not change this result. Preferential RNP staining and deoxyribonucleic acid (DNA) specific staining failed to reveal the thin filaments interconnecting the nucleolar granules. We suggest that the latter filaments serve as support for pre-rRNA processing and for storage of large pre-ribosomal subunits.

Animals↗

PubMed2001

Antigen processing for MHC class I restricted presentation of exogenous influenza A virus nucleoprotein by B-lymphoblastoid cells.

In general, exogenous proteins are processed by antigen-presenting cells in the endosomes for major histocompatibility complex (MHC) class II presentation to CD4+ T cells, while proteins synthesized endogenously are processed in the cytoplasm for MHC class I presentation to CD8+ T cells. However, it is recognized that exogenous proteins can be processed for MHC class I presentation also, and evidence in favour of alternatives to the conventional MHC class I processing and presentation pathway is accumulating. Here, we show that exogenous recombinant influenza A virus nucleoprotein (rNP) is processed for MHC class I presentation to CD8+ cytotoxic T lymphocytes (CTL) by EBV-transformed, B-lymphoblastoid cell lines (B-LCL). Processing of rNP for HLA-B27-associated presentation seemed to follow the conventional MHC class I pathway predominantly, as presentation was diminished in the presence of lactacystin and brefeldin A, but was less sensitive to chloroquine and NH4Cl. HLA-B27-associated presentation was also observed using cells lacking a functional transporter associated with antigen processing, suggesting that alternative pathways may be exploited for processing of rNP.

Acetylcysteine↗

PubMed1981

Structural organization of nascent transcripts and hnRNA molecules in amphibian oocytes.

Comparisons of relative lengths of lampbrush loops, nascent RNP transcripts and hnRNA molecules from oocytes of amphibia with different C-values show that there is an increasing trend in loop, and transcriptional unit, length with increase in genome size but no increasing trend with respect to RNA contour length. The formation of duplex regions and circles in RNP fibrils indicates that RNA processing may occur within the nascent fibrils. The hnRNA molecules from oocytes of the various amphibia readily form intermolecular duplex structures. These complementary sequences have a low kinetic complexity and are transcribed from highly repetitive sequences distributed throughout the genome. Their possible function is considered.

Animals↗

PubMed1995

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli.

Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.

Amino Acid Sequence↗

PubMed1977

[Transcription complexes in subnuclear fractions isolated from mammalian cells: ultrastructural study].

Miller Beatty's technique was adapted to the study of definite chromatin fractions (nucleolar and nonnucleolar chromatin) isolated from Mammalian cells. The ultrastructural organization of the transcriptional complexes obtained depended on the nuclear compartment studied. In isolated nucleoli, there were "Christmas-tree"-like figures. In nonnucleolar chromatin, the figures were different from the former by the internal structure of the RNP fibrils being synthesized.

Animals↗

PubMed2001

Packing and delivery of a genetic message.

Balbiani rings (BRs) 1 and 2 are two exceptionally large chromosomal puffs on chromosome IV in the salivary glands of the dipteran Chironomus tentans. The BR genes are 35-40 kb, contain four short introns, and encode salivary polypeptides of one million molecular weight. They have proven uniquely suited for visualization of the assembly and transport of a specific messenger ribonucleoprotein (RNP) particle. A BR transcript is packed with proteins into a thin RNP fibril, which is folded into a compact ring-like structure. The completed BR particle is released from the gene and moves randomly in the nucleoplasm before it becomes associated with the nuclear pore complex. The passage through the nuclear pore is a highly ordered process with a series of consecutive steps: initial binding, docking, unfolding, movement through the pore with the 5' end of the transcript in the lead, and exit into the cytoplasm. On the cytoplasmic side, the RNA becomes immediately engaged in protein synthesis. Recently, several major proteins in the BR particle have been identified and characterized. They are added to the BR RNA molecule concomitantly with transcription. During the ensuing RNA transport, the various proteins behave differently, some remaining in the nucleus, others entering the cytoplasm coupled to the RNA. The flow pattern of a given protein seems to be closely related to the specific function of the protein. The RNA-binding proteins are likely to play various active roles during gene expression rather than being solely packaging proteins. Finally, it is emphasized that the co-transcriptional loading of the transcript with proteins is probably a key process in gene expression that to a large extent determines the fate of an mRNA both in the nucleus and the cytoplasm.

Animals↗

PubMed1999

Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase.

BACKGROUND: Colicins are antibiotic-like proteins of Escherichia coli that kill related strains. Colicin E3 acts as an RNase that specifically cleaves 16S rRNA, thereby inactivating the ribosomes in the infected cell. The producing organism is protected against colicin E3 by a specific inhibitor, the immunity protein Im3, which forms a tight 1:1 complex with colicin E3 and renders it inactive. Crystallographic studies on colicin E3 and Im3 have been undertaken to unravel the structural basis for the ribonucleolytic activity and its inhibition. RESULTS: The crystal structure of Im3 has been determined to a resolution of 1.8 A. The structure consists of a four-standard antiparallel beta sheet flanked by three alpha helices on one side of the sheet. Thr7, Phe9, Phe16 and Phe74 form a hydrophobic cluster on the surface of the protein in the vicinity of Cys47. This cluster is part of a putative binding pocket which also includes nine polar residues. CONCLUSIONS: The putative binding pocket of Im3 is the probable site of interaction with colicin E3. The six acidic residues in the pocket may interact with some of the numerous basic residues of colicin E3. The involvement of hydrophobic moieties in the binding is consistent with the observation that the tight complex can only be dissociated by denaturation. The structure of Im3 resembles those of certain nucleic acid binding proteins, in particular domain II of topoisomerase I and RNA-binding proteins that contain the ribonucleoprotein (RNP) sequence motif. This observation suggests that Im3 has a nucleic acid binding function in addition to binding colicin E3.

Bacterial Proteins↗

PubMed1992

Antibodies to carbonic anhydrase in systemic lupus erythematosus and other rheumatic diseases.

OBJECTIVE: Autoantibodies to CA were demonstrated in the sera of patients with systemic lupus erythematosus (SLE) and some other rheumatic diseases. This study was undertaken to define the isoform and species specificity of these reactions, as well as to develop a method for detecting immune complexes. METHODS: Antibodies to CA were sought by enzyme-linked immunosorbent assay (ELISA) and by Western immunoblotting. RESULTS: An increased prevalence of CA autoantibodies was detected, by both methods, in patients with SLE, scleroderma, and polymyositis, compared with controls. In SLE patients, CA autoantibodies occurred preferentially in those with anti-U1 RNP or anti-U1 RNP and Ro/SS-A. Some sera reacted with only the CA I or CA II isoform, while approximately 50% of sera that were CA positive reacted with both isoforms. The autoantibodies reacted preferentially with the human enzymes, rather than the bovine CA, both on Western blot and by ELISA: Selected IgG F(ab')2 fragments from anti-CA-containing sera specifically inhibited the enzyme activity of CA, and the CA inhibitor acetazolamide partially inhibited the binding of anti-CA to CA. Thus, at least a part of autoanti-CA is directed toward the active site of CA. Finally, CA molecules were detected as immune complexes in sera from selected anti-CA-positive patients. CONCLUSION: Autoantibodies to CA represent a previously unrecognized autoantibody to an abundant intracellular protein of the human erythrocyte.

Acetazolamide↗

PubMed1989

C-reactive protein reacts with the U1 small nuclear ribonucleoprotein.

C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.

Antigen-Antibody Reactions↗

PubMed1975

[Transcription complexes with nucleolar and chromosomal origins in oocytes of Pleurodeles waltlii and P. poireti (amphibia, urodela) (author's transl)].

The method of spreading transcription complexes has been applied to amphibian oocytes of Pleurodeles genus. Complexes of nucleolar origin show a regular and homogeneous organization similar to that described in other materials. The observations add to the interpretation as an amplification of nucleolar DNA and a redundancy of ribosomal cistrons in the two species studied. -- On the other hand, complexes of chromosomal origin display a great diversity. Two main characteristics can be drawn: the existence of several transcription units in a chromosomal organization unit and the possibility to point out a special architecture at the RNP fibril level. Applying a shadowing technique used for isolated molecules is an improvement compared with earlier methods based on PTA coloration.

Amphibians↗

PubMed1980

Chromatin organization during meiotic prophase of Bombyx mori.

Chromatin organization during the early stages of male meiotic prophase in Bombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200-300 A in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5-25 mu of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.

Animals↗

PubMed1988

Chemical cross-linking of Sm and RNP antigenic proteins.

Nuclear extracts, competent for in vitro premessenger RNA splicing, were chemically cross-linked with thiol-reversible reagents in order to study the organization of proteins within ribonucleoprotein particles (RNPs) containing uridine-rich small nuclear RNAs (UsnRNPs). The distribution of select UsnRNP antigens within cross-linked complexes was determined by Western blotting of diagonal two-dimensional gels. On the basis of calculations from the molecular weights of cross-linked complexes containing UsnRNP common proteins B', B, and D, it is proposed that each of these proteins was associated with UsnRNP common proteins E and G. In addition, D' is proposed to be positioned close to D. The spatial distribution of UsnRNP common proteins was such that B' and B could not be cross-linked to D. The data also suggested that the 63-kDa U1 snRNP specific protein was cross-linked to other U1-specific proteins, particularly C, but not to the UsnRNP common proteins. We propose that part of the UsnRNP core of common proteins contains at least two asymmetrical copies of B':B:D:D':E:G with stoichiometries of 2:1:1:1:1:1 and 1:2:1:1:1:1.

Cell Nucleus↗

PubMed1996

A role for ribosomal protein L5 in the nuclear import of 5S rRNA in Xenopus oocytes.

Prior to ribosome assembly, 5S ribosomal RNA (5S rRNA) binds to ribosomal protein L5 to form a stable ribonucleoprotein particle (5S RNP). We have analyzed the role of L5 binding in the nuclear targeting of 5S rRNA in Xenopus oocytes, and have compared the nuclear import pathway of 5S RNPs with other karyophilic molecules. Nuclear import of in vitro-generated 5S RNPs was found to be sensitive to three general inhibitors of nuclear pore complex-mediated translocation: ATP depletion, chilling, and wheat germ agglutinin. The initial rate and extent of net nuclear import was threefold greater with preassembled 5S RNPs than with 5S rRNA microinjected alone, suggesting that L5 binding is a prerequisite for nuclear accumulation. Nuclear import of 5S rRNA/5S RNPs is a facilitated process dependent on limiting factors, since nuclear import exhibited saturation kinetics. Not only was nuclear import of labeled 5S rRNA reduced in the presence of excess unlabeled 5S rRNA, but also in the presence of the synthetic karyophilic protein P(lys)-BSA. In contrast, import was not inhibited by U1 small nuclear RNA (snRNA) or U3 small nucleolar RNA (snoRNA). 5S rRNA/5S RNP nuclear import therefore appears to follow a pathway of molecular interactions similar to many karyophilic proteins, but not the methylguanosine cap-dependent U1 snRNA pathway or the cap-independent U3 snoRNA pathway.

Animals↗

PubMed2003

Downregulation of eNOS mRNA expression by TNFalpha: identification and functional characterization of RNA-protein interactions in the 3'UTR.

OBJECTIVE: We have previously shown that downregulation of endothelial nitric oxide synthase (eNOS) expression by tumour necrosis factor-alpha (TNF alpha) resulted entirely from the marked destabilization of the eNOS mRNA. As the 3'-untranslated region (3'UTR) in many eukaryotic mRNA has been well documented to bind regulatory trans-factors in the control of transcript stability, we have examined protein binding to this region of the eNOS mRNA. A high degree of homology amongst human and bovine 3'UTR also suggests that important functional features that are conserved through evolution are present within this region. METHODS: RNA-protein interactions were studied in cross-linking assays, in which radiolabelled RNA encoding the human eNOS 3'UTR or selected sequences was incubated with cytoplasmic extracts of cultured human umbilical vein endothelial cells (HUVECs). Serial 5'- and 3'-truncated deletional mutations of the eNOS 3'UTR were generated to identify the specific binding sequences. eNOS mRNA expression in HUVECs was assessed by RT-PCR analysis. RESULTS: Using radiolabelled RNA encoding the entire 418-nucleotide 3'UTR, we have identified ribonucleoprotein complexes (RNPs) of approximate molecular weights of 53, 56 and 66 kDa in the endothelial extracts. The formation of the 53- and 56-kDa RNPs was upregulated by TNF alpha, while the formation of the 66-kDa RNP was downregulated. Formation of the 53-kDa RNP was favoured by RNA fragments that contained sequences from the proximal and distal portions of the 3'UTR, whereas the formation of the 66-kDa RNP was favoured by RNA fragments with the AU-rich distal end. RNA fragments containing a CU-rich 158-nucleotide sequence from the medial portion of the eNOS 3'UTR (designated M158) favoured the formation of the 56-kDa RNP. Adenoviral gene transfer and overexpression of M158 RNA, as a protein-binding decoy to prevent the formation of the 56-kDa RNP on the endogenous transcripts, attenuated the TNF alpha-induced downregulation of eNOS mRNA in cultured endothelial cells. CONCLUSION: Our results demonstrate that the regulation of eNOS expression involves the specific binding of cytoplasmic proteins to highly conserved elements along the 3'UTR, and the 56-kDa RNP represents a novel regulatory trans-factor in the destabilization of eNOS transcripts.

3' Untranslated Regions↗

PubMed1996

Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts.

The product of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K (U1-70K) gene, a U1 snRNP-specific protein, has been implicated in basic as well as alternative splicing of pre-mRNAs in animals. Here, we report the isolation of full-length cDNAs and the corresponding genomic clone encoding a U1-70K protein from a plant system. The Arabidopsis U1-70K protein is encoded by a single gene, which is located on chromosome 3. Several lines of evidence indicate that two distinct transcripts (short and long) are produced from the same gene by alternative splicing of the U1-70K pre-mRNA. The alternative splicing involves inclusion or exclusion of a region (910 bp) that we named "included intron." Two transcripts were clearly detectable in all tissues tested, and the level of the transcripts varied in different organs. The deduced amino acid (427 residues) sequence from the short transcript has strong homology to the animal U1-70K protein and contains an RNA recognition motif, a glycine hinge, and an arginine-rich region characteristic of the animal U1-70K protein. The long transcript has an in-frame translational termination codon within the 910-bp included intron, resulting in a truncated protein containing only 204 amino acids. The protein encoded by the short transcript is recognized by U1 RNP-specific monoclonal antibodies and binds specifically to the Arabidopsis U1 snRNA, whereas the protein from the long transcript does not. In addition, multiple polyadenylation sites were observed in the 3' untranslated region. These results suggest a complex post-transcriptional regulation of Arabidopsis U1-70K gene expression.

Alternative Splicing↗

PubMed1995

Crystal structure of the U1A spliceosomal protein complexed with its cognate RNA hairpin.

We have determined the crystal structure of the RNA binding domain of the U1A spliceosomal protein bound to a 21-nucleotide RNA hairpin at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the four-stranded beta-sheet of the RNP domain as an open structure. The AUUGCAC hexanucleotide sequence interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. The stacking interaction between RNA bases and aromatic protein side chains and the extensive hydrogen bonding network involving RNA bases, protein side chains and protein mainchain amide and carbonyl groups are crucial for the sequence specific recognition of RNA.

Base Sequence↗

PubMed1980

The CREST syndrome: a distinct serologic entity with anticentromere antibodies.

The CREST syndrome is a variant of systemic sclerosis characterized by the presence of calcinosis. Raynaud's phenomenon, esophageal motility abnormalities, sclerodactyly and telangiectasia. The serums of 27 patients with the CREST syndrome have been examined for the presence of antinuclear antibodies. Twenty-six of 27 (98 percent) serums contained high titers (> one:80) of an antibody that produces a discrete speckled pattern of immunofluorescence on a human laryngeal carcinoma cell line (HEp-2). The antibody has been shown to react with the centromeric region of metaphase chromosomes. This antibody was also found in three of 14 patients with Raynaud's disease, in one of 60 patients with systemic lupus erythematosus, in three of 26 patients with systemic sclerosis with diffuse scleroderma and in one of 15 patients with mixed connective tissue disease. The antibody was not detected in the serums of patients with rheumatoid arthritis. Sjögren's sicca complex or linear scleroderma. Patients with osteoarthritis who were age- and sex-matched to the group with the CREST syndrome did not have anticentromere antibodies. Autoantibodies found in other connective tissue diseases (anti-DNA, anti-RNP, Sjögren's syndrome antigen B (anti-SS-B) were not found in serums from patients with the CREST syndrome. A case report illustrating the appearance of the anticentromere antibody at a time when Raynaud's phenomenon antedated the clinical diagnosis of CREST syndrome is presented.

Adult↗