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M Caizergues-Ferrer

Publications and source records attributed to M Caizergues-Ferrer.

At least 37 records · Page 2Linked to original sources
PubMed1994

U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

Base Sequence↗
PubMed1994

Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase.

The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5' external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.

Animals↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1992

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↗
PubMed1991

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis.

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.

Animals↗
PubMed1990

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↗
PubMed1990

Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin.

Amino Acid Sequence↗
PubMed1989

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin.

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.

Amino Acid Sequence↗
PubMed1989

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↗
PubMed1988

A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1.

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N-terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer. We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high-mobility-group type.

Amino Acid Sequence↗
PubMed1987

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↗
PubMed1985

Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells.

Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector lambda gt11, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (beta gal-cDNA encoded protein) was developed to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90% of a full length cDNA. By probing a Northern blot with the 100 kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.

Amino Acid Sequence↗
PubMed1984

Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells.

The synthesis of preribosomal RNA is inhibited "in vivo" and "in vitro" by the protease inhibitor leupeptin. "In vivo" leupeptin decreases by 74% the incorporation of labeled uridine into 45S pre rRNA while the synthesis of other RNA species is only slightly decreased. "In vitro", the elongation of already initiated pre rRNA chains that is achieved by incubation of isolated nucleoli is blocked by leupeptin. On the other hand, "in vitro" leupeptin has no direct effect on RNA polymerase I, tested in a nonspecific transcriptional system with Calf thymus DNA as template and in run off experiments with a cloned DNA containing the initiation site of the rDNA gene. A 100 kDa nucleolar protein which has been shown to be endoproteolytic cleaved "in vivo" (1) acts as an inhibitor of rDNA transcription in presence of leupeptin but produces little effect on the nonspecific transcription. In absence of the drug, the 100 kDa protein is processed in specific peptides which appeared to be similar to the "in vivo" maturation products. The possible role of the 100 kDa maturation process in the regulation of rDNA transcription is discussed.

Animals↗
PubMed1984

Induction of new proteins in the nuclear matrix of CHO cells by a heat shock: detection of a specific set in the nucleolar matrix.

An early response to an incubation of CHO cells for one hour at 43 degrees C is the appearance in the nuclear matrix of new proteins: 2 proteins (48 and 46 kDa) were associated with the extranucleolar part of the nuclear matrix, 6 other proteins (130, 100, 95, 85, 76 and 70 kDa) were present in matrices prepared from isolated nucleoli. In addition, a large increase in the amount of associated minor components is observed. It is suggested that these early modifications in composition of the matrix are related to the inhibition of rRNA and hnRNA synthesis and maturation.

Animals↗
PubMed1983

Histones from Chinese hamster ovary cells. Multiple modified forms: a quantitative approach.

A quantitative analysis of histone variants and of their modified forms in exponentially growing Chinese hamster ovary cells is described. A two-step analytical procedure has been developed. Total nuclear proteins were first fractionated on a sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. After electroelution from the corresponding bands cut out of the gel, each individual histone was analysed on an acetic acid/urea/Triton X-100 slab gel. All the variants and most of the acetylated and phosphorylated molecules were thus resolved. In absence of butyrate, we have detected four, three and two different levels of acetylation respectively for H4, H3, H2A and H2B. Scanning of the stained gel has allowed the determination of the relative amount of each forms. H3 and H4 present a higher turnover of the acetate groups than H2A and H2B. For the four histones, acetate label appeared preferentially in the multi-acetylated forms then gradually in the less acetylated forms. The data suggest that the deacetylation-acetylation process of a given histone molecule involves all the acetate groups of the molecule (three for a triacetylated histone, one for a monoacetylated histone).

Acetylation↗