Claude Brunel

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells: Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs☆

Abstract U 1 snRNP † isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U 1 molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U 1 core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle. When a less drastically purified U 1 snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected. When a mixture of U 1 , U 2 , U 4 , U 5 and U 6 snRNPs, which contains the same four/five polypeptides as U 1 snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U 2 , U 4 and U 5 were found in addition to those derived from U 1 . No fragment derived from U 6 was found. In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U) n G with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U 2 , where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U 6 snRNA, it does not contain the sequence A(U) n G which correlates well with the fact that no U 6 snRNA fragment seems to resist micrococcal nuclease digestion.

Activation and inhibition processes of alkaline phosphatase from bovine brain by metal ions (Mg2+ and Zn2)

Abstract 1. 1 Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC3.1.3.1) from bovine brain is strongly activated by Mg2+ (5–4-fold). In this respect the enzymes behaves like that of kidney or bone marrow but is different from the intestinal and placental enzymes which are much less activated. 2. 2 The rate of the activation process is pH-dependent. At pH 10.0, the pH optimum of p nitrophenyl phosphate hydrolysis, the activation process is rapid. At lower pH values, it is slow and can be described by an exponential relationship vs time. The results suggest that the activation by Mg2+ proceeds through a binding of the metal with free enzyme which might be followed by a conformational change, rather than by an action on the substrate. 3. 3 Other metal ions can also bind to the brain enzyme. Mn2+, Co2+, Ni2+ with an activating effect, Zn2+ with an inhibitory effect. They all bind to the enzyme at the same site and also induce the supposed conformational change. However, Zn2+ is able to produce interactions, perhaps with Pi which is one of the products of hydrolysis, which might explain its inhibitory effect.

Genomic imprinting controls matrix attachment regions in the Igf2 gene.

ABSTRACT Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.

H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation

H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.

Imidazole: an inhibitor of L-phenylalanine-insensitive alkaline phosphatases of tissues other than intestine and placenta.

Abstract 1. 1. Alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from brain, kidney, liver, bone, lung and spleen, which are not very sensitive to l -phenylalanine, are strongly inhibited by imidazole, whereas the placental and intestinal enzymes, which are very sensitive to l -phenylalanine, are only slightly affected. This is a new possibility for distinguishing the alkaline phosphatase isoenzymes. 2. 2. The inhibition is apparently of an uncompetitive type, suggesting that the inhibitor interacts with the ES complex to form an EIS complex. 3. 3. Histidine acts upon all enzymes assayed in this study. Its inhibition is of a mixed type. 4. 4. An interpretation, taking into account the role of the zinc atom(s) present in the active center, is offered.

hnRNP from HeLa cells contain a ribonuclease active on double-stranded RNA

Abstract A ribonuclease D, i-e acting against double-stranded RNA structures like poly r(AU), was identified in ribonucleoprotein structures containing the heterogenous nuclear RNA (hnRNP) from HeLa cells. This activity could not however be detected in intact hnRNP but only after passage through a DEAE-cellulose column or digestion by a combination of ribonucleases A+T1. This enzyme does not degrade poly r(A)-poly d(T) nor poly r(A), nor does it yield mononucleotides, excluding the possibility of a non-specific exonuclease type of activity like phosphodiesterase. It is inhibited by ethidium bromide and double-stranded RNA and resembles in all respects so far investigated the ribonuclease D previously isolated from Krebs cells by Rech et al (Nucl. Acids Res. 1976, 3, 2055–2065).

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 (Brunelet 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 Uinf1sup* described by Lerneret 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.

L'activité pyrophosphatasique de la phosphatase alcaline du cerveau

Resume 1. 1. The pyrophosphatase activity of alkaline phosphatase from bovine brain depends on Mg2+ bound to the enzyme, rather than on the formation of the complex ion, MgP2O72−. Indeed, the Mg2+-enzyme (the enzyme preincubated with Mg2+) hydrolyses the Mg2+-free pyrophosphate and its activity reaches a maximum value without Mg2+ added to the assay medium. The Mg2+-free enzyme hydrolyses the MgP2O72− complex, but a weaker activity is observed. Its activity towards P2O74− only reaches 1–5% of the value obtained with the Mg2+-enzyme. 2. 2. When present at relatively high concentration, MgP2O72− is not hydrolyzed, but is able to activate the rate of p-nitrophenyl phosphate hydrolysis at pH 8.5, indicating that by its binding, MgP2O72− has the same activating effect as Mg2+. 3. 3. Among divalent metal ions which can bind to the enzyme, Mg2+ is the most effective in inducing the pyrophosphatase activity. Mn2+, Ni2+, Co2+ and particularly Zn2+ have weaker effects. 4. 4. It is concluded that the true substrate of the pyrophosphatase activity of alkaline phosphatases is P2O74−, which behaves like a phosphomonoester.

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 desease. 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 polypettide with a molecular weight of about 15 000 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 be different from the snRNPs described by Lerner et al.

Phosphatase alcaline du cerveau de boeuf Role du magnésium sur l'action de la l-phénylalanine et d'autres acides amines

Abstract 1. 1. Alkaline phosphatase from beef brain (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) is not very sensitive to l -phenylalanine. The stereospecific effect is only 38%, compared to 65% and 90% for the calf intestinal and human placental enzymes, respectively. For all three enzymes, the phenomenon is uncompetitive and not allosteric in nature. 2. 2. At pH d -isomer activates the brain phosphatase and has no effect on the two other enzymes. l -Phenylalanine is also an activator with respect to the control and its stereospecific effect is only evident when compared with the d -isomer. 3. 3. This activation is closely related to the activation by Mg2+. Both phenomena are pH dependent, practically identical from pH 8.5 to 10.0 and their effect on the enzyme is noncompetitive. Mg2+ is a possible site for the binding of l -phenylalanine to the brain enzyme. 4. 4. All the natural α-amino acids have an identical behaviour. The d -isomers produce the same activation as glycine, whereas the effect of l -isomers, themselves activators when compared with the control, is dependent on the molecular weight of the amino acid (alanine, serine have no effect, maximum effect is observed with tryptophan and on its conformation (the effects are different with leucine, isoleucine, norleucine). For the intestinal and placental phosphatases, only l -phenylalanine and l -tryptophan have an important effect. 5. 5. The mode of action of phenylalanine and the other amino acids capable of causing a stereospecific effect on the brain enzymes seems to be different from the one described previously in the literature for the rat intestinal and human placental alkaline phosphatases. It is suggested that the strong or the weak sensitivity, of these two kinds of enzymes, to Mg2+ may be responsible for the observed differences. The role of Mg2+ on the reactivity of alkaline phosphatases is also discussed.