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J Stevenin

Publications and source records attributed to J Stevenin.

At least 19 recordsLinked to original sources
PubMed2006

An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.

A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.

Amino Acid Motifs↗
PubMed2001

UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation.

Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s). In the presence of TBP-free TAF(II) complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.

Acetylation↗
PubMed2001

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation.

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Alternative Splicing↗
PubMed2000

Alternative splicing of intron 3 of the serine/arginine-rich protein 9G8 gene. Identification of flanking exonic splicing enhancers and involvement of 9G8 as a trans-acting factor.

9G8 protein belongs to the conserved serine/arginine-rich (SR) protein family, whose members exhibit multiple functions in constitutive and alternative splicing. We have previously shown that 9G8 primary transcripts are subjected to alternative splicing by excision/retention of intron 3 and to a tissue specific modulation. Because both 5'- and 3'-splice sites of intron 3 appear to be suboptimal in vertebrates, we tested the 9G8 intron 3 as a novel model system of alternative splicing. By using an in vitro approach and a mutational analysis, we have identified two purine-rich exonic splicing enhancers (ESE) located in exon 4 and a (GAA)(3) enhancer located in exon 3. These elements act in concert to promote efficient splicing activation both in vitro and in vivo. Titration experiments with an excess of exonic enhancers or SR-specific RNA targets strongly suggest that SR proteins are specifically involved in the activation process. Although ASF/SF2 was expected to interact the most efficiently with ESE according to the enhancer sequences, UV cross-linking coupled or not to immunopurification demonstrates that 9G8 is highly recruited by the three ESE, followed by SC35. In contrast, ASF/SF2 only binds significantly to the (GAA)(3) motif. S100 complementation experiments with individual SR proteins demonstrate that only 9G8 is able to fully restore splicing of intron 3. These results, and the fact that the exon 3 and 4 ESE sequences are conserved in vertebrates, strongly suggest that the alternative splicing of intron 3 represents an important step in the regulation of the expression of 9G8.

Alternative Splicing↗
PubMed1999

A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities.

An important group of splicing factors involved in constitutive and alternative splicing contain an arginine/serine (RS)-rich domain. We have previously demonstrated the existence of such factors in plants and report now on a new family of splicing factors (termed the RSZ family) from Arabidopsis thaliana which additionally harbor a Zn knuckle motif similar to the human splicing factor 9G8. Although only around 20 kDa in size, members of this family possess a multi-domain structure. In addition to the N-terminal RNA recognition motif (RRM), a Zn finger motif of the CCHC-type is inserted in an RGG-rich region; all three motifs are known to contribute to RNA binding. The C-terminal domain has a characteristic repeated structure which is very arginine-rich and centered around an SP dipeptide. One member of this family, atRSZp22, has been shown to be a phosphoprotein with properties similar to SR proteins. Furthermore, atRSZp22 was able to complement efficiently splicing deficient mammalian S100 as well as h9G8-depleted extracts. RNA binding assays to selected RNA sequences indicate an RNA binding specificity similar to the human splicing factors 9G8 and SRp20. Taken together, these result show that atRSZp22 is a true plant splicing factor which combines structural and functional features of both h9G8 and hSRp20.

Alternative Splicing↗
PubMed1999

Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation.

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

Adenovirus E1A Proteins↗
PubMed1993

Contrasted cis-acting effects of downstream 5' splice sites on the splicing of a retained intron: the adenoviral E1A pre-mRNA model.

The adenoviral E1A pre-mRNA contains an upstream intron (the 216 nucleotide intron) which is spliced only weakly both in vivo and in vitro. We have chosen the E1A transcript as a model to analyse, in vitro, the role of downstream cis-elements involved in the alternative splicing of this retained intron. By using a series of constructs containing specific deletions, mutations and/or truncations, we show that the 13S 5' splice site, positioned 259 nucleotides downstream of the 216 nucleotide intron, is the main cis-element which activates the splicing of this intron. Our results establish the importance of a downstream 5' splice site for the activation of the 3' splice site, which is known to be suboptimal within this retained intron. Unexpectedly, the 12S 5' splice site, although positioned at an ideal distance (121 nucleotides) from the upstream intron, does not exhibit such a cis-acting effect. In contrast, its improvement to a consensus sequence may even result in a slight negative cis-acting effect in the presence of the 13S 5' splice site, which is the first observation of such a feature. We have shown that this unexpected behaviour is due, at least partly, to the unusual characteristics of the wild-type upstream intron, which requires a hairpin structure between the branch sites and the 3' splice site to reduce the operational distance between these two sites. Possible mechanisms involved in the contrasted cis-acting effects of the 13S and 12S 5' splice sites are discussed.

Adenovirus E1A Proteins↗
PubMed1989

A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

We have characterized a Mediterranean beta-thalassemia allele containing a sequence change at codon 30 that alters both beta-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G----C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg----Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

Adult↗
PubMed1989

The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

Adenoviridae↗
PubMed1988

In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site.

During the analysis of the in vitro alternative splicing of the natural E1A transcript of adenovirus, other minor reactions were detected (Schmitt et al., 1987, Cell 50, 31-39). We report here their characterization. The first reaction concerns the excision of a 216 nucleotide intron delineated by the 9S 5' splice site and a 3' splice site 216 nucleotides downstream. It can occur on the premRNA transcript and the 13S and 12S mRNA species. Strikingly, the reaction uses one of 3 branch points located 51, 55 or 59 residues upstream of the 3' splice site, a distance which is unusually long since all the branch points mapped up to now are located between 18-37 nucleotides of the 3' splice site. The dramatic accumulation of the corresponding lariat intermediates, likely related to this long spacing indicates that the second splicing step is relatively unefficient. The second kind of reaction analysed is a cryptic splicing which uses a 3' splice site generated by the junction of the 13S mRNA exons, and leads to the formation of psi 12S and psi 9S mRNAs. In vitro, this reaction occurs only from a 13S mRNA transcript, and not from the 13S mRNA newly formed in the splicing assay, consistent with what has been observed in vivo. Thus, both the well known alternative and the minor reactions occurring in vivo from E1A premRNA and mRNAs are detected in vitro, implying that most of the alternative splicing machinery is reconstituted in the in vitro system.

Adenovirus Early Proteins↗
PubMed1980

Comparison of the nuclear ribonucleoproteins containing the transcripts of adenovirus-2 and HeLa cell dna.

A method as devised allowing the preparation of hnRNA-containing ribonucleproteins (hnRNP) frm HeLa cells infected with adenovirus type 2 under conditions where the extraction of viral replication complexes as minimal. Approximately 60% of the RNA from such hnRNP hybridized with adenovirus DNA. The hnRNP from infected cells had the same general characteristics as those from uninfected cells. Their size was heterogeneous (30-260 S) and depended upon that of their RNA. Their CsCl densities were identical (1.39-1.40 g/ml), indicating the same protein:RNA ratio. Their proteins were found in the same molecular weight range, between 25 000 and 200 000. The major proteins of hnRNP from HeLa cells were present in hnRNP from adenovirus-infected cells. As 60% of the cellular RNA was replaced by adenovirus RNA in hnRNP, this in dicated that there was not stringent specificity in the RNA-protein interactions. The relative proportions of the proteins were identifical in both cases, suggesting that the hnRNP assembly was independent of nucleotide sequences at least for the major proteins. The hnRNP from infected and uninfected cells differed by the size of their RNA, which was larger after infection, and by the presence of six additional minor polypeptides after infection. However, it cannot be excluded that the presence of these polypeptides in hnRNP resulted from non-specific adsorption.

Adenoviruses, Human↗
PubMed1977

Complexity of the structure of particles containing heterogeneous nuclear RNA as demonstrated by ribonuclease treatment.

Brain ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) and a large number of polypeptides ranging from 23000 to 150000 molecular weight were treated with pancreatic and T1 ribonucleases at low (0.1 or 0.5 microng plus 5 or 25 units/ml), high (2 microng plus 100 units/ml) and very high (20 microng plus 1000 units/ml) concentrations. At low enzyme concentration, a large fraction of the particle material accumulated at 35-45 S. The accumulation was more marked for proteins in the 30000-38000 molecular weight range. Heterogeneous complexes containing a large spectrum of proteins with a characteristic distribution were disclosed between 60 and 200 S. At high ribonuclease concentration the total quantity of proteins at 35-45 S decreased, but the proteins of 30000-38000 molecular weight predominated. Heterogeneous complexes were also present. Finally at very high enzyme concentration, the particles were almost entirely hydrolyzed. Only a small amount of heterogeneous complexes subsisted at 30-190 S. The RNA content of the remaining ribonucleoprotein complexes as well as its size decreased upon ribonuclease treatment as shown by CsCl density determination and electrophoretic analysis. The results suggested that the stability of the complexes depended upon protein-protein as well as RNA-protein interactions. They also showed that sequences up to 200-300 nucleotides were protected by proteins against ribonuclease. The results were compatible with the existence of at least 3 constituents in the particles. The previously defined monoparticle population accumulating at 35-45 S was heterogeneous in respect to ribonuclease sensitivity and protein composition. The 30000-38000 molecular weight proteins accumulating at low ribonuclease and predominating afterwards were assumed to belong to monoparticles alpha. Monoparticles beta contained a larger range of proteins more easily released by the enzymes. The heterogeneous complexes were a third constituent whose relationship with the monoparticles war not established yet. A large fraction of the particle phosphoproteins was associated to these complexes. On the basis of our experiments in vitro it is assumed that monoparticle alpha can be preferentially isolated from the nuclei under conditions where endogeneous ribonuclease is high and (or) not inhibited. This might explain why, in certain cases, only one or a few proteins were described in nuclear particles instead of the complete set present in the native particles.

Animals↗
PubMed1977

Protease activities during preparation and handling of nuclear particles containing hnRNA.

Conditions were devised to avoid protease activity during the preparation and the subsequent handling of nuclear particles containing hnRNA. During all the steps of preparation of rat liver particles, the presence of phenylmethyl sulfonyl fluoride (PMSF) was required for the reproducibility of the results. It probably inhibited the cellular serine proteases before the separation of the particles from the other cellular structures. Protease activity was detected in the rat liver particles. The enzyme(s) preferentially hydrolyzed a few particle polypeptides. It was not inhibited by PMSF, nor by two trypsin and chymotrypsin-like protease inhibitors, nor by iodoacetamide, but was inhibited by sodium bisulfite and para-hydroxymercury benzoate (PHMB). PHMB was preferred above bisulfite because it could be used at lower concentration. It proved useful when particles were to be incubated at 37 degrees C. A protease hydrolysing the same polypeptides as the liver enzyme was also detected in rat brain particles. However, its activity was much lower in this tissue and the presence of protease inhibitors was not absolutely required under the standard conditions of preparation and handling of brain particles.

Animals↗
PubMed1977

Metrizamide dissociates nuclear particles containing heterogeneous nuclear RNA.

Metrizamide gradients were tested for the possible fractionation of the constitutive units of nuclear particles. Material from 35-55 S monoparticles was indeed distributed along the gradient but rerun experiments, CsCl density determinations, formaldehyde fixation prior to centrifugation suggested that the separation was due to dissociation or (and) action of endogeneous ribonucleases rather than to monoparticle fractionation. That dissociation had indeed occured was confirmed by the study of 60-110 S polyparticles. They were dissociated into ribonucleoproteins rich in phosphoproteins and into free proteins. These products were essentially similar to those obtained after NaCl treatment of the particles though the modes of action of metrizamide and NaCl are likely to be different. The loss of proteins from particles reaches 60-70% and we conclude that metrizamide gradients are not utilizable for the fractionation of the units of nuclear particles.

Animals↗