The "frost sign" and the "snow white sign": intramucosal air injection or peroxide colitis?
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Air↗
Biomedical subjects
B Lapeyre
Publications and source records attributed to B Lapeyre.
Duration of esophagogastroduodenoscopy procedures.
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Procedural Sedation↗
The two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p.
We report here the characterization of a bypass suppressor of pab1Delta which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1Delta, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.
Amino Acid Sequence↗
Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro.
We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [(3)H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.
Alleles↗
Gar1p binds to the small nucleolar RNAs snR10 and snR30 in vitro through a nontypical RNA binding element.
The nucleolar proteins Gar1p and fibrillarin possess a typical nucleolar glycine/arginine-rich domain and belong to ribonucleoprotein particles. Both proteins are essential for yeast cell growth and are required for pre-rRNA processing. In addition, Gar1p is involved in pre-rRNA pseudouridylation, whereas fibrillarin is required for pre-rRNA methylation. Gar1p and fibrillarin are each associated with a different subset of the small nucleolar RNAs (snoRNAs). Gar1p is co-immunoprecipitated with the H/ACA family of snoRNAs, whereas fibrillarin is co-immunoprecipitated with the C/D family. However, attempts to demonstrate direct interactions between fibrillarin and snoRNAs have failed, and such interactions between Gar1p and the H/ACA snoRNAs had not yet been reported. Among the H/ACA snoRNAs associated with Gar1p, one can distinguish a large group of snoRNAs that are not essential in yeast and serve as guides for pseudouridine synthesis onto the pre-rRNA molecule. In contrast, the two snoRNAs snR10 and snR30 are required for normal cell growth and for pre-rRNA cleavage. We show here that Gar1p interacts in vitro directly and specifically with these two snoRNAs. Deletion analysis of Gar1p indicates that a major RNA binding element, which is extremely well conserved throughout evolution, lies in the middle of the protein. However, this domain alone binds poorly to the target RNAs and an accessory domain is required to restore efficient binding. The accessory domain can be either one of the glycine/arginine-rich domains or a second element of the core of the protein that is highly conserved between different species.
Amino Acid Sequence↗
Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant.
mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8 (YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that the spb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3' end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3'-5' mRNA degradation intermediates in yeast.
Amino Acid Sequence↗
gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.
Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.
Amino Acid Sequence↗
Identification of a segment of the small nucleolar ribonucleoprotein-associated protein GAR1 that is sufficient for nucleolar accumulation.
GAR1 is a 25-kDa nucleolar protein that is essential for yeast cell growth. The protein is associated with a subset of small nucleolar RNAs and is required for pre-rRNA processing. By expressing in yeast various deletions of GAR1 fused to a reporter protein, we have searched for which particular domain of GAR1 can account for its nucleolar localization. We report here that the glycine/arginine-rich domains of GAR1, which are shared by several other nucleolar proteins, are neither sufficient nor required for the steady-state accumulation of the fusion protein in the nucleolus. We further demonstrate that the central domain of GAR1 is both sufficient to target the beta-galactosidase to the yeast nucleolus and to restore the growth of a strain deficient in GAR1. As opposed to the other characterized nucleolar proteins, the nucleolar targeting domain of GAR1 does not exhibit any homology with the SV40 T-antigen-type nuclear localization sequence. Moreover, none of the modified GAR1 proteins that we examined has allowed us to distinguish the nuclear and nucleolar targeting domains. The presence in GAR1 of a single domain that is responsible for both nuclear entry and nucleolar accumulation suggests that GAR1 either could be carried piggyback by another nucleolar component, possibly as part of a small nucleolar ribonucleoprotein particle, or could be transported to the nucleolus by using a pathway different from the other nucleolar proteins.
Amino Acid Sequence↗
Nucleotide sequence of the Schizosaccharomyces pombe 25S ribosomal RNA and its phylogenetic implications.
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Animals↗
The SpGAR1 gene of Schizosaccharomyces pombe encodes the functional homologue of the snoRNP protein GAR1 of Saccharomyces cerevisiae.
GAR1 is a nucleolar protein which is associated with small nucleolar RNAs (snoRNAs) and which is required for pre-ribosomal RNA processing. In Saccharomyces cerevisiae, the GAR1 gene is essential for cell viability. We have cloned and sequenced the GAR1 gene from the distantly related yeast Schizosaccharomyces pombe. The SpGAR1 gene, which contains two small introns, codes for a 194 amino-acid protein of 20 kDa. A protein sequence comparison indicates that SpGAR1 is 65% identical to ScGAR1. Anti-ScGAR1 antibodies recognize SpGAR1, emphasizing the structural conservation of the protein. Immunostaining of S.pombe cells with these antibodies reveals that SpGAR1 is localized in the nucleolus, as is the case in S.cerevisiae. Moreover, SpGAR1 can substitute for GAR1 in S.cerevisiae, indicating that the two proteins are functionally equivalent. These results suggest a parallel evolutionary conservation of proteins and RNAs with which GAR1 interacts in mediating its pre-rRNA processing and viability functions. After fibrillarin, GAR1 is the second protein of the snoRNPs shown to have been conserved throughout evolution.
Amino Acid Sequence↗
Study of multiple fibrillarin mRNAs reveals that 3' end formation in Schizosaccharomyces pombe is sensitive to cold shock.
Fibrillarin is a nucleolar protein which is associated with small nucleolar RNAs, and is required for pre-rRNA processing. We have cloned and characterized the gene encoding fibrillarin in the fission yeast Schizosaccharomyces pombe and we have followed its expression under various conditions. Fission yeast fibrillarin is a 305 amino-acid protein which appears to be highly conserved throughout evolution. In Xenopus, human or Saccharomyces cerevisiae, a single fibrillarin mRNA is detected while, in S. pombe a single copy gene encodes different mRNAs which differ at the 3' ends. Under normal growth conditions, two mRNAs of 1.1 and 1.35 kb are detected with the 1.1 kb being the most abundant. Both the total amount and relative abundance of these two mRNAs are strongly affected by exposure to low temperature, namely the 1.1 kb mRNA almost disappears while the 1.35 kb is less markedly diminished. A new species of 3.2 kb accumulates in the cell, which contains an unusually long 3' untranslated region of 2 kb. We have found that exposure of the cells to a cold shock has a profound effect on 3' end formation in S.pombe since the transcription of several other mRNAs is also capable of skipping the normal 3' end site to terminate at a further downstream site.
Amino Acid Sequence↗
The amino terminus of mammalian nucleolin specifically recognizes SV40 T-antigen type nuclear localization sequences.
Nucleolin is a major nucleolar protein in mammalian cells that is thought to be involved in ribosome biogenesis. The discovery that nucleolin shuttles between the cytoplasm and the nucleus raises the possibility that it is also involved in transporting ribosomal or nuclear proteins to the nucleus. The three structural domains of nucleolin bear a striking resemblance to the domains of a previously identified yeast protein NSR1, although the two proteins do not share a high degree of sequence similarity. NSR1 specifically recognizes the nuclear localization sequence (NLS) of both the simian virus large T antigen (SV40 T-antigen) and the yeast histone H2B by ligand blot analysis, and is a candidate for a receptor involved in the initial stages of nuclear transport. We report here that nucleolin, either purified from Chinese hamster ovary (CHO) cells or expressed in yeast, also specifically recognizes the wild-type, but not a mutant, histone H2B nuclear localization sequence by ligand blot analysis. The NLS recognition site is located within the N-terminal domain of both proteins. In showing that nucleolin, a protein that moves between the cytoplasm and the nucleus, also has the ability to interact with nuclear localization signals, our data support the idea that shuttling nucleolar proteins play a role in nuclear transport.
Amino Acid Sequence↗
GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast.
Among the few proteins of the eukaryotic nucleolus that have been characterized, four proteins, nucleolin, fibrillarin, SSB1 and NSR1, possess a common structural motif, the GAR domain, which is rich in glycine and arginine residues. In order to examine whether the presence of this domain is characteristic of a family of nucleolar proteins, we investigated whether other yeast genes encode proteins containing GAR domains. We report here the sequence and the characterization of a new yeast gene, GAR1, which encodes a protein of 205 residues containing two GAR domains. GAR1 is a non-ribosomal protein, localized in the yeast nucleolus, which is essential for cell growth. Immunoprecipitation with anti-GAR1 antibodies shows that GAR1 is associated with a subset of snoRNAs, including snR10 and snR30. Depletion of GAR1 by expression under the control of a regulated GAL promoter, impairs processing of the 35S primary transcript of pre-rRNA and prevents synthesis of 18S rRNA. GAR1 is thus the fifth member of a family of nucleolar proteins containing GAR domains, and is involved in rRNA metabolism.
Amino Acid Sequence↗
Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast.
NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.
Amino Acid Sequence↗
Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease.
Autoantibodies against U3 small nuclear ribonucleoprotein are associated with scleroderma autoimmune disease. They were shown to react with fibrillarin, a 34- to 36-kilodalton protein that has been detected in all eukaryotes tested from humans to yeasts. We isolated a 1.6-kilobase cDNA encoding fibrillarin from a Xenopus laevis cDNA library. The protein contains a 79-residue-long Gly-Arg-rich domain in its N-terminal region and a putative RNA-binding domain with ribonucleoprotein consensus sequence in its central portion. This is the first report of cloning of fibrillarin, and the deduced protein sequence is in agreement with the involvement of the protein in a ribonucleoprotein particle.
Amino Acid Sequence↗
Nucleolin from Xenopus laevis: cDNA cloning and expression during development.
Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis.
Amino Acid Sequence↗
Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons.
Nucleolin is a multifunctional nucleolar protein involved in the synthesis, packaging and maturation of pre-rRNA in eukaryotic cells. We describe the molecular organization and complete sequence of the mouse nucleolin gene, the first higher eukaryotic gene encoding a protein that is both an RNA binding protein involved in rRNA processing and a specific nucleolar protein. The nucleolin gene extends over 9000 base-pairs and is split into 14 exons that encode the 706 amino acid residues of the protein. The promoter sequence is G + C-rich (67% G + C) with four G/C boxes, it lacks bona fide TATA and CAAT boxes and shows capping site heterogeneity. The existence of pyrimidine-rich motifs, similar to those found in the promoter of ribosomal protein genes, could be relevant to the co-regulation of genes whose products are involved in ribosome biogenesis. Nucleolin contains four RNA binding domains, each about 80 amino acid residues long, which include the 11-residue core ribonucleoprotein consensus motif. Each domain is encoded by two exons, with an intervening sequence interrupting the conserved core motif at roughly the same amino acid position. This latter result suggests that the RNA binding domains are composed of two independent subdomains, whose functions remain to be determined.
Amino Acid Sequence↗
Phosphorylation of nucleolin by a nucleolar type NII protein kinase.
Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.
Amino Acid Sequence↗