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C Brunel

Publications and source records attributed to C Brunel.

At least 55 records · Page 3Linked to original sources
PubMed1986

A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein.

Using a protein blotting method for the detection of nucleic acid binding proteins, we have identified in HeLa cell nuclear extracts an intron binding protein (IBP) that selectively recognizes the 3' splice site region of mammalian pre-mRNAs. The binding site was accurately delineated using oligonucleotides complementary to human beta-globin pre-mRNA. It spans the 3' splice site AG dinucleotide and the crucial polypyrimidine stretch upstream, but includes neither the branchpoint nor the lariat structure. Although the technique used here shows that the binding specificity is an intrinsic property of IBP and does not depend on snRNA-pre-mRNA interactions, it comigrates with U5 snRNP and is immunoprecipitated by anti-Sm antibody. This strongly suggests that IBP belongs to U5 snRNP. We propose that it is involved in one of the earliest steps of the splicing reaction by mediating the interaction of U5 snRNP with the 3' splice site.

Base Sequence↗
PubMed1986

A human auto-immune antibody specifically recognizing initiator methionine tRNA from yeast and higher eucaryotes.

Analysis of sera from 168 patients with autoimmune disorders revealed that one patient with Sjôgren's syndrome produced antibodies against deproteinized initiator methionine tRNA in addition to those against La protein. This anti-tRNAimet recognizes also tRNAimet from yeast but not from Phaseolus vulgaris chloroplasts (bean) or E. coli. It appears therefore that the epitope could be located in the TF loop in which an A residue in position 60 and the AUCG sequence are the only common features in yeast and human tRNAimet.

Autoantibodies↗
PubMed1986

RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells.

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.

Base Sequence↗
PubMed1984

Mg2+ induces a sharp and reversible transition in U1 and U2 small nuclear ribonucleoprotein configurations.

When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, 1983), were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (less than or equal to 5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162: 623-643, 1982). At high Mg2+ (greater than or equal to 7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.

Base Sequence↗
PubMed1983

Interaction of snRNAs with rapidly sedimenting nuclear sub-structures (hnRNPs) from HeLa cells.

We have shown previously (Liautard et al., 1982, J. Mol. Biol., 162, 623-643) that digestion with micrococcal nuclease under drastic conditions of a pure U1 snRNP, as well as a mixture containing U2, U1, U4, U5 and U6 snRNPs, gives rise to resistant RNA fragments derived from all but U6 snRNAs. As an attempt to elucidate the way in which snRNPs are attached to their native structure, the same approach was applied to hnRNP which are known to contain snRNP (Guimont-Ducamp et al., 1977, Biochimie, 59, 755-758). Micrococcal nuclease digestion of hnRNPs yielded a population of 15-50 nucleotides long resistant fragments of snRNAs. Sequence analyses showed that all fragments previously identified in core snRNPs were also present. Only U2 and U5 snRNAs were further protected as a result of their association with the hnRNP complex (from the cap to nucleotide 32 for U2 and from nucleotide 22 to nucleotide 70 for U5). No additional protected fragment derived from U1, U4 and U6 snRNAs was found. This finding confirms that the 5' terminal region of U1 snRNP remains available for base-pairing interaction with the premessenger RNA, as predicted by the model of Lerner et al. (Nature, 1980, 283, 220-224).

Base Sequence↗
PubMed1982

Mouse DNA sequences complementary to small nuclear RNA U1.

A mouse genomic library was screened for sequences complementary to U1 nuclear RNA. Out of the eight clones tested, none contained more than one copy of U1. Six of them were identical and one of those (clone 0U1-XIII) was further analyzed. This latter clone contained no other gene for discrete species of small size RNA in the 8 Kb EcoRI fragment encoding U1. A 248 bp Bg1II fragment from 0U1-XIII encompassing the full length of U1 as well as flanking regions on both sides has been subcloned and sequenced in M13 phage. Although the coding region was 96.5% homologous to rat U1a RNA, there is no direct evidence that this clone is a true gene. 3' and 5' flanking sequences of this as well as other published clones have been searched for homologies and the results of this search are discussed.

Animals↗
PubMed1982

Relationship between snRNA species contained in nuclear antigens recognized by autoantibodies and the clinical profile in systemic rheumatic diseases.

Antibodies to extractable nuclear antigens (ENA) are generally used in the diagnosis of connective tissue diseases. Using a rapid, very sensitive method we have shown that extractable nuclear antigens, which are now well-characterized at the molecular level, differ by their RNA content. The method was applied to the sera of 17 patients suffering from different connective tissue diseases. The results show that mixed connective tissue disease (MCTD) and other mild connective tissue diseases are characterized by the presence in the antigen of U1 small nuclear RNA (U1 snRNA) only. On the other hand, antibodies from 6 out of 8 patients tested with Systemic Lupus Erythematosus (SLE) recognize antigens exhibiting a more complex RNA pattern. Three of them precipitated all five snRNAs U2, U1, U4, U5, U6 whereas some snRNAs were lacking or quantitatively less important in precipitates obtained with the three others.

Antibodies, Antinuclear↗
PubMed1981

Primary structure identification of snRNAs present in highly purified snRNPs from HeLa cells.

Extensive purification of snRNPs as a subset of hnRNP from HeLa cells has been previously reported (Brunel et al. (1981), Nucleic Acids Research, 9, 815). These snRNPs were shown to contain discrete RNA species comigrating in gel electrophoresis with authentic U1, U2, U4, U5 and U6 species. We now report sequence analysis data of about 50 nucleotides from the 3'-end which serve to positively establish the identity of snRNAs present in these purified snRNPs. Sequence heterogeneity was found at the 3'-end of U4 species. A minor species identical to U1 at its 3'-end but slightly shorter was identified as the U1 described by Lerner et al. (Nature (1980) 283, 220-224) through sequencing of the 5'-end. When unfixed hnRNP are centrifuged in a CsCl gradient containing 4M guanidinium chloride instead of 0.5% sarkosyl as above, a band containing only one RNA species was observed. T1 RNAse fingerprinting and sequence analysis of the oligonucleotides produced allowed identification of this RNA as U5 snRNA.

Base Sequence↗
PubMed1981

Particles containing small molecular weight nuclear RNAs (snRNPs). Structure and possible functions.

Recent knowledge on snRNPs is reviewed in this paper. The relevant findings of our laboratory were essentially as follows: Particles containing small nuclear RNAs (snRNAs) were characterized ten years ago. More recently Lerner et al. have shown that particles containing snRNAs react with antibody produced in autoimmune disease. Furthermore, the snRNA (some of them are probably involved in 'splicing') were found associated with hnRNP. In the present work we have studied structures, extracted from hnRNP that contain snRNAs. We were able to obtain and purify ribonucleoproteins complexes containing some of the snRNAs. These particles (snRNPs) are very stable. They were purified by three different successive cycles of centrifugation under denaturing conditions. The particles are characterized by a density of 1,43 g/cm3 in CsCl and a sedimentation coefficient of 11--12S. They contain five species of snRNAs (U1, U2, U4, (U5), U6 according to the nomenclature of Lerner et al.) and at least one polypeptide with a molecular weight of about 15000 daltons. An other particle containing only U5 was also isolated. These snRNPs are not disaggregated in media destabilizing ionic forces, hydrophobic interaction or hydrogens bonds and seem to different from the snRNPs described by Lerner et al.

Animals↗
PubMed1981

Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells.

A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220-224) were found in these purified snRNPs, while a significant part of U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cytoplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.

Base Sequence↗
PubMed1979

Protein kinase associated with hnRNP from Hela cell nuclei. Partial purification and properties.

The protein kinase previously described as an endogenous activity present in ribonucleoprotein particles containing heterogenous RNA from HeLa cells (Blanchard et al, Eur. J. Biochem (1977), 79, 11-131) has been partially purified by a combination of chromatography on DEAE cellulose, phosphocellulose and Sephacryl S-200. It is able to phosphorylate exogenous substrates, among which casein is the most efficient. Its enzymatic properties were found to be quite similar to those described for the endogenous activity. Its activity is independent of cyclic AMP as well as of the calcium-dependent regulator protein and is inhibited by hemin. Its native molecular weight is around 48,000 as determined by gel filtration.

Calcium↗