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P Fabrizio

Publications and source records attributed to P Fabrizio.

35 records · Page 2Linked to original sources
PubMed1997

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p.

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.

Amino Acid Sequence↗
PubMed1996

The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.

Amino Acid Sequence↗
PubMed1996

Conferring drug resistance by MDR1 gene transfection increases susceptibility to irradiation and lipid peroxidation in 3T3 cell line.

This study was performed to test the hypothesis that conferring multiple drug resistance reduces cell susceptibility to irradiation and iron-stimulated lipid peroxidation. Multidrug resistant (PN1A) and parental drug sensitive (PSI-2) cell lines were exposed to ADP-Fe or Ascorbate-Fe complexes at 37 degrees C and to irradiation. Lipid peroxidation was estimated by the TBA test, whereas x-ray effect was estimated by clonogenic assay. Cell glutathione-S-transferase (GST), total and Se-dependent glutathione peroxidase (GSH-Px) activities, and glutathione and vitamin E were measured. PN1A produced more peroxides than PSI-2 after exposure to iron complexes and formed fewer colonies after irradiation. Higher activities of GST and total and Se-GSH-Px were observed in PN1A. Vitamin E and total glutathione did not differ in the two cell subclones. These data show that the induction of the mdr1 phenotype by transfection of mdr1 gene in 3T3 cells increases susceptibility to irradiation and iron stimulated lipid peroxidation.

3T3 Cells↗
PubMed1995

Isolation and characterization of the small nucleolar ribonucleoprotein particle snR30 from Saccharomyces cerevisiae.

The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1. All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation. NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the small nucleolar ribonucleoprotein particle snR30 (snoRNP). In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein. Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure. The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients. Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide.

Base Sequence↗
PubMed1995

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.

Amino Acid Sequence↗
PubMed1995

Susceptibility to lipid peroxidation of human hepatocellular carcinoma cell lines with different levels of multiple drug-resistant phenotype.

BACKGROUND: It has not been established whether the presence of intrinsic or acquired multiple drug-resistant (MDR) phenotype affects susceptibility to undergo iron-stimulated lipid peroxidation. EXPERIMENTAL DESIGN: To assess this point, human hepatocellular carcinoma cell lines with moderate, clinically relevant (P1) or elevated (P1(0.5)) MDR phenotype and their parental drug-sensitive (P5) cell line were exposed to ADP-Fe or ascorbate-Fe complexes and H2O2 in different experiments. Thiobarbituric acid-reactive substances (TBARS) were measured. Total cell glutathione, glutathione-S-transferase, total and selenium-dependent glutathione peroxidase activities, and cell alpha-tocopherol content were also determined. RESULTS: P5 and P1 cell lines showed similar and significant formation of TBAR after 1-hour incubation exposure to iron complexes, whereas P1(0.5) subclone did not. No accumulation of TBAR was observed during the exposure to H2O2 in the three cell lines. Among antioxidants, only alpha-tocopherol cell content was significantly higher in P1(0.5) in comparison with either P1 or P5. CONCLUSIONS: These data suggest that MDR phenotype development per se does not increase resistance to iron-related free radical attack in human hepatocellular carcinoma cell lines. Resistance to undergo lipid peroxidation is associated only to high degrees of drug resistance and appears more related to increased alpha-tocopherol cell content rather than an MDR phenotype.

Adenosine Diphosphate↗
PubMed1994

Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts.

Small nuclear ribonucleoprotein (snRNP) particles are essential for pre-messenger RNA splicing. In human HeLa cells, 40 proteins associated with snRNPs have been identified. Yet, the function of many of these proteins remains unknown. Here, the immunoaffinity purification of the spliceosomal snRNPs U1, U2, U4/U6.U5, and several nucleolar snRNP species from the yeast Saccharomyces cerevisiae is presented. The U1 and U4/U6.U5 snRNPs were purified extensively and their protein composition and ultrastructure analyzed. The yeast U1 snRNP is larger and contains three times more specific proteins than its human counterpart. In contrast, the size, protein composition, and morphology of the yeast and the human U4/U6.U5 snRNPs are significantly similar. The preparative isolation of yeast snRNPs will allow the cloning as well as genetic and phylogenetic analysis of snRNP proteins which will accelerate our understanding of their function.

Blotting, Western↗
PubMed1993

Gas-containing lumbar disc herniation. A case report and review of the literature.

The finding of gas into the intervertebral disc space ("vacuum phenomenon") is relatively common, whereas gas-containing cervical or lumbar disc herniations are rarely observed. A case of a patient with bilateral leg pain, provoked by a computed tomography scan and surgically documented L4-L5 gas-containing disc herniation is reported and the cases previously described in detail are reviewed, to define the pathophysiologic and diagnostic aspects of this rare phenomenon associated with a herniated disc fragment.

Female↗
PubMed1992

Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro.

A thorough mutational analysis of U6 RNA in combination with a functional reconstitution assay, revealed that three domains are important for U6 function in pre-mRNA splicing. In order to further analyze why these regions are so critical for splicing, we make use of phosphorothioate substituted U6 RNAs. Wild-type U6 RNA was transcribed in vitro with T7 RNA polymerase in the presence of either phosphorothiate (alpha-S) ATP, GTP, UTP or CTP. The functionality of the transcripts was monitored by in vitro reconstitution. While substitution with alpha-S ATP, GTP or UTP blocked splicing, substitution with alpha-S CTP had little or no effect on splicing. We made use of this alpha-S CTP effect in an attempt to elucidate which phosphates in the U6 RNA molecule play a role in the first or in the second step of splicing. U6 mutants in which a change of an A, G or U to C does not have any significant effect on splicing were transcribed in the presence of alpha-S CTP. Observed effects on splicing thus have to be attributed to the presence of the thio-substituted phosphate group rather than the nucleotide change. The results of in vitro reconstitution give a clear answer for at least three phosphates; two of them play a role in the first step, while one of them is involved in the second step of splicing.

Base Sequence↗
PubMed1990

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing.

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.

Base Sequence↗
PubMed1989

In vitro reconstitution of functional yeast U2 snRNPs.

A system for the functional reconstitution of yeast U2 snRNPs using synthetic U2 RNAs is described. We use oligonucleotide-directed RNase H cleavage to specifically deplete yeast extracts of their endogenous full-length U2 snRNA and consequently inactivate pre-mRNA splicing activity. The subsequent addition of synthetic yeast U2 RNAs, derived by in vitro transcription (T7U2 RNAs), to these oligonucleotide-treated extracts efficiently reconstitutes their ability to splice pre-mRNA. The use of deletion derivatives of the T7U2 RNA has demonstrated that the region downstream from the conserved Sm-binding site sequence in the yeast U2 RNA is not absolutely required for pre-mRNA splicing activity in vitro. Furthermore, we found that both human and rat U2 RNAs can function in yeast extracts. We also show that point mutations in the yeast U2 RNA can be analyzed using the in vitro reconstitution system. Allele-specific suppression of mutations in pre-mRNA branch site sequence is observed when the appropriate compensatory mutations in the branch site recognition region of the T7U2 RNA are introduced. Finally, we present a model for the interaction of the U2 and U6 snRNAs during pre-mRNA splicing.

Base Sequence↗
PubMed1989

In vitro assembly of yeast U6 snRNP: a functional assay.

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.

Base Sequence↗
PubMed1987

Comparative mutational analysis of wild-type and stretched tRNA3(Leu) gene promoters.

We demonstrate that, when the yeast tRNA(3Leu) gene is stretched so that the distance between the two portions of the intragenic promoter is increased to 365 base pairs, the A and B blocks remain functional. Mutations in the A block, which show a weak phenotype when inserted in the wild type, exert a dramatic effect when inserted into the stretched gene. Experiments with extensively purified transcription factor tau indicate that the tau B-B block interaction is not influenced by A-B distance; only the ability of tau A to interact with A block sequences is affected, possibly because of the additional free-energy cost of forming a large loop of the intervening DNA.

Base Sequence↗
PubMed1980

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus.

Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.

Animals↗