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PubMed1991

Intramolecular base pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.

Animals↗

PubMed1991

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.

Antisense Elements (Genetics)↗

PubMed1992

A novel gene, spp91-1, suppresses the splicing defect and the pre-mRNA nuclear export in the prp9-1 mutant.

Processing and export of nuclear pre-mRNA are believed to be competing processes in the nucleus. In order to identify factors which are involved in these processes, we isolated suppressors that relieve the growth defect of a prp9-1 temperature-sensitive mutant strain of Saccharomyces cerevisiae. The prp9-1 mutation was previously shown to abolish splicing and to target pre-mRNA to the cytoplasm. One of the suppressors, spp91-1, corrects the prp9-1 growth defect through partial restoration of splicing and by a complete reversion of the pre-mRNA escape phenotype. This suppressor is specific for two prp9 alleles and cannot substitute for PRP9 function. The mutant and wild-type alleles of SPP91 were cloned and sequenced. SPP91 encodes a novel protein essential for mitotic growth whose sequence contains motifs indicative of a nuclear localization. In vivo depletion of SPP91 in a prp9-1 genetic background is lethal and is associated with reduced amounts of spliced mRNA and accumulation of pre-mRNA. This observation strongly supports the hypothesis that SPP91 encodes a PRP factor. We suggest that spp91-1 increases pre-mRNA retention in the nucleus by improving the formation of the spliceosome and thereby allowing a larger proportion of the pre-mRNA molecules to be spliced.

Alleles↗

PubMed1993

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.

Animals↗

PubMed1994

Two major tertiary folding transitions of the Tetrahymena catalytic RNA.

The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.

Animals↗

PubMed1994

NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.

Amino Acid Sequence↗

PubMed1994

Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs.

Translation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-protein interactions between iron-responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal protein U1A and mRNAs which harbour specific binding sites for this protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor protein-mediated inhibition of 43S pre-initiation complex binding.

5-Aminolevulinate Synthetase↗

PubMed1995

Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins.

The SDB23 gene of Saccharomyces cerevisiae was isolated in a search for high copy-number suppressors of mutations in a cell cycle gene, DBF2, SDB23 encodes a 21,276 Da protein with significant sequence similarity to characterized mammalian snRNP core proteins. Examination of multiple sequence alignments of snRNP core proteins with Sdb23p indicates that all of these proteins share a number of highly conserved residues, and identifies a novel motif for snRNP core proteins. Sdb23p is essential for cell viability and is required for nuclear pre-mRNA splicing both in vivo and in vitro. Extracts prepared from Sdb23p-depleted cells are unable to support splicing and have vastly reduced levels of U6 snRNA. The stability of U1, U2, U4 and U5 spliceosomal snRNAs is not affected by the loss of Sdb23p. Antibodies raised against Sdb23p strongly coimmunoprecipitate free U6 snRNA and U4/U6 base-paired snRNAs. These results establish that SDB23 encodes a novel U6 snRNA-associated protein that is essential for the stability of U6 snRNA. We therefore propose the more logical name USS1 (U-Six SnRNP) for this gene.

Amino Acid Sequence↗

PubMed2003

Infectious mononucleosis patients temporarily recognize a unique, cross-reactive epitope of Epstein-Barr virus nuclear antigen-1.

The spectrum of antibodies against Epstein-Barr virus nuclear antigen-1 (EBNA-1) in patients with a recent history of infectious mononucleosis and nonaffected EBV-positive individuals has been characterized by epitope mapping. Sera were evaluated for antibodies to all unique maximally overlapping octapeptides of EBNA-1. Both normal controls and patients with infectious mononucleosis produce IgG antibodies that recognize the glycine-alanine-rich portion of EBNA-1, as previously described. All EBNA-1 IgG-positive infectious mononucleosis patients tested, however, consistently produce IgG specific for an additional epitope (aa 398-412 PPPGRRPFFHPVGEA) near the middle of the EBNA-1 protein. This region was not found to be antigenic in healthy EBV-seropositive individuals. This region does, however, cross-react with the sequence PPPGMRPP from the common lupus spliceosomal autoantigen Sm B' in several infectious mononucleosis patient sera. Patients with recent clinical infectious mononucleosis temporarily recognize a unique cross-reactive epitope of EBNA-1 not bound by antibodies from non-infectious mononucleosis EBV-positive sera or those with a distant history of IM.

Amino Acid Sequence↗

PubMed2003

Myocytes positive for in situ markers for DNA breaks in human hearts which are hypertrophic, but neither failed nor dilated: a manifestation of cardiac hypertrophy rather than failure.

The significance of DNA breaks reported in failing hearts is controversial, although they may suggest myocyte apoptosis and may thus be responsible for the progression of heart failure. This study attempted to check the validity of the in situ markers for DNA breaks for detecting myocyte death and to evaluate separately two factors, failure or hypertrophy, crucial for DNA breaks in pathological human hearts. In the autopsy study, myocytes showed positivity for in situ nick end-labelling (TUNEL) and of Taq and Pfu polymerase-based in situ ligation assays not only in dilated cardiomyopathy (DCM, n = 9) with failure, but also in hypertrophic cardiomyopathy (HCM, n = 8) and hypertensive heart disease (HHD, n = 4) without failure. There was a significant correlation between each in situ marker and heart weight. The incidence of TUNEL-positive myocytes always exceeded that seen in in situ ligation assays. In addition, there were significant correlations between the in situ markers and the expression of the proliferating cell nuclear antigen (PCNA) and of the spliceosome component of 35 kD (SC-35). Similarly, in the left ventricular biopsy study using 23 DCM, 21 HCM, 11 HHD, and 13 non-hypertrophic hearts, the incidence of the in situ markers showed significant correlations with the left ventricular mass index and myocyte size, but not with cardiac function and dilatation. Positivity of myocytes for in situ markers for DNA breaks, such as TUNEL and in situ ligation assays, may be an epiphenomenon accompanying cardiac hypertrophy, but not myocyte death in pathological human hearts.

Adult↗

PubMed2001

A nuclear-encoded intein in the fungal pathogen Cryptococcus neoformans.

We have used comparative sequence analysis to identify an intein-like sequence (protein splicing element) present in Cryptococcus neoformans, a fungal pathogen of humans. The sequence encoding this element is present in the C. neoformans PRP8 gene, as an in-frame insertion relative to the PRP8 genes of other organisms. It contains sequences similar to those of the protein-splicing domains of two previously described yeast inteins (in Saccharomyces cerevisiae and Candida tropicalis), although it lacks any recognizable internal endonuclease domain. The Cryptococcus neoformans intein (Cne PRP8) is only the second to be found in a eukaryote nuclear genome; the previously described yeast inteins occur at the same site in the VMA gene homologues of S. cerevisiae and C. tropicalis. The host gene of the Cryptococcus intein, PRP8, encodes a highly conserved mRNA splicing protein found as part of the spliceosome. The Cne PRP8 intein may be a useful drug target in addressing the cryptococcal infections so prevalent in AIDS patients.

Amino Acid Sequence↗

PubMed1994

Regulation of alternative splicing of the fibronectin IIICS domain by cytokines.

The primary fibronectin gene transcript is alternatively spliced in three regions, designated EIIIA, EIIIB and IIICS. While the functions of the EIIIA and EIIIB domains are still to be established, either end of the IIICS domain contains the cell-binding sites CS1 and CS5 recognised by the integrin VLA-4 which is present on mononuclear cells. We have determined the effects of three cytokines upon fibronectin mRNA and IIICS splice variants in four cultured human cell lines using Northern hybridisation and PCR. In the cell line CAKI-2, interleukin 1 beta, Platelet-derived growth factor BB and transforming growth factor beta 1 all increased mRNA splice variants where the CS1 and CS5 regions were removed. This cytokine-induced change would lead to a decrease in the CS1 and CS5 cell binding sites within the extracellular matrix. All the changes in spliceosome function increase use of downstream 3' splice acceptor sites and may represent use of a single common pathway by three different cytokines.

Alternative Splicing↗

PubMed1995

Galectin-3 is a nuclear matrix protein which binds RNA.

The endogenous galectin-3 is a carbohydrate-binding protein of M(r) approximately 30,000 serving in the cytoplasm and on the cell surface as a receptor for ligands containing poly-N-acetyllactosamine sequences. In addition, galectin-3 has been demonstrated to be associated in the nucleus with ribonucleoprotein complexes and to act as a pre-mRNA splicing factor and to be involved in spliceosome assembly. However, little is known about either its nuclear localization or its ligand(s), respectively. We demonstrate directly here that galectin-3 is associated with the RNA protein skeleton of the nucleus, i.e., the nuclear matrix, and binds with single-stranded DNA (ssDNA) and with RNA. The affinity of binding was determined to be 2.3 microM. Lactose, which inhibits galectin-3 binding to glycoconjugates, failed to inhibit either galectin-3-ssDNA or galectin-3-RNA binding. Galectin-3 exhibited the highest affinity to poly(A) ribonucleotide homopolymers. The results presented here shows that galectin-3 may act as a RNA-binding protein in the nuclear matrix in a non-carbohydrate-dependent manner.

Animals↗

PubMed1999

SRPK1 and LBR protein kinases show identical substrate specificities.

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing and the lamin B receptor (LBR), an integral protein of the inner nuclear membrane, are among the best characterized proteins that contain RS domains. Two SR Protein-specific Kinases, SRPK1 and SRPK2, have been shown to phosphorylate specifically the RS motifs of the SR family of splicing factors and play an important role in regulating both the spliceosome assembly and their intranuclear distribution, whereas an LBR-associated kinase, that specifically phosphorylates a stretch of RS repeats located at the NH2-terminal region of LBR, has been recently purified and characterized from turkey erythrocyte nuclear envelopes. Using synthetic peptides representing different regions of LBR and recombinant proteins produced in bacteria we now demonstrate that SRPK1 modifies LBR with similar kinetics and on the same sites as the LBR kinase, that are also phosphorylated in vivo. These data provide significant evidence for a new role of SRPK1 in addition to that of pre-mRNA splicing.

Amino Acid Sequence↗

PubMed2001

Lupus autoantibodies recognize the product of an alternative open reading frame of SmB/B'.

An unusual feature of the gene for the spliceosomal protein SmB/B' is the presence of an unusually long alternative open reading frame (aORF) which could encode 220 amino acids. We cloned and expressed this aORF protein and used immunological assays to determine its antigenicity in patients with systemic lupus. Sera from 10 of 22 (46%) anti-Sm positive lupus patients showed significant binding to the SmB' aORF protein by ELISA while neither the normal controls nor anti-Sm negative lupus patient controls showed significant reactivity. Antigenicity of the SmB' aORF protein was further localized to the C-terminus using a deletion construct. This is the first known example in which the product of an alternative open reading frame acts as an autoantigen in human disease. These results are consistent with the possibility that generation of anti-Sm autoantibodies in a subset of lupus patients is due to abnormal processing and expression of an aORF SmB/B' message, by an as yet unidentified mechanism.

Adult↗

PubMed1993

Intranuclear distribution of poly(A) RNA determined by electron microscope in situ hybridization.

We used a biotinylated poly(dT) probe to localize poly(A) RNA in HeLa cells at optical and electron microscope levels. We established that the fluorescent speckled staining pattern corresponds at the ultrastructural level to the labeling of perichromatin fibrils, at least part of the population of perichromatin granules, and clusters of interchromatin granules. Coiled bodies and the interchromatin granule-associated zones, a recently described subcompartment containing U1 but not U2 snRNA, were not labeled. The density of the labeling of interchromatin granule clusters exceeded by three to five times that of the surrounding extranucleolar area. These results are discussed in relation to the role of perichromatin fibrils in splicing of pre-mRNA and to the possible involvement of interchromatin granules in the assembly of mature spliceosomes as well as in sorting and/or coordination of RNA molecules to be transported to the cytoplasm.

Cell Compartmentation↗

PubMed1997

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Antigens, Nuclear↗

PubMed1997

Nuclear accumulation of the U1 snRNP-specific protein C is due to diffusion and retention in the nucleus.

The U1 small nuclear ribonucleoprotein particle (snRNP) has an important function in the early formation of the spliceosome, the multicomponent complex in which pre-mRNA splicing takes place. The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. Here we show by microinjection of Xenopus laevis oocytes that the third U1 snRNP-specific protein, U1C, passively enters the nucleus. Furthermore, we show that in both X. laevis oocytes and cultured HeLa cells mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP.

Animals↗