Biomedical subjects

J Stévenin

Publications and source records attributed to J Stévenin.

At least 19 recordsLinked to original sources

PubMed2003

Remodelling of the host cell RNA splicing machinery during an adenovirus infection.

Adenovirus makes extensive use of RNA splicing to produce a complex set of spliced mRNAs during virus replication. All transcription units, except pIX and IVa2, encode multiple alternatively spliced mRNAs. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the viral life cycle are produced. The complex interaction between host cell RNA splicing factors and viral regulatory elements has been studied intensely during the last decade. Such studies have begun to produce a picture of how adenovirus remodels the host cell RNA splicing machinery to orchestrate the shift from the early to the late profile of viral mRNA accumulation. Recent progress has to a large extent focused on the mechanisms regulating E1A and L1 alternative splicing. Here we will review the current knowledge of cis-acting sequence element, trans-acting factors and mechanisms controlling E1A and L1 alternative splicing.

Adenoviridae Infections↗

PubMed2001

TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs.

TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.

Alternative Splicing↗

PubMed2001

SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs.

SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3' untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a significant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3' untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, specifically activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the first example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.

Alternative Splicing↗

PubMed2000

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection.

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Animals↗

PubMed2000

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site.

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

Animals↗

PubMed1999

The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers.

The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.

Alternative Splicing↗

PubMed1998

Characterization of SRp46, a novel human SR splicing factor encoded by a PR264/SC35 retropseudogene.

The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

3T3 Cells↗

PubMed1997

The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.

Alternative Splicing↗

PubMed1997

Cloning of human 2H9 heterogeneous nuclear ribonucleoproteins. Relation with splicing and early heat shock-induced splicing arrest.

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45 degrees C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H' family. Protein 2H9 displays two consensus sequence-type RNA binding domains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H'. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

Amino Acid Sequence↗

PubMed1996

The mouse poly(C)-binding protein exists in multiple isoforms and interacts with several RNA-binding proteins.

The murine poly(C)-binding protein (mCBP) was previously shown to belong to the group of K-homology (KH) proteins by virtue of its homology to hnRNP-K. We have isolated cDNA-splice variants of mCBP which differ by two variable regions of 93 bp and/or 39 +/- 3 bp respectively. Both variable regions are located between the second and third KH-domain of mCBP. The characterization of a partial genomic clone enabled us to propose a model for the generation of the second variable region by the use of a putative alternative splice signal. The mCBP mRNA is expressed ubiquitously and the protein is found predominantly in the nucleus with the exception of the nucleoli. We have identified five proteins which interact with mCBP in the yeast two hybrid system: mouse y-box protein 1 (msy-1), y-box-binding protein, hnRNP-L, filamin and splicing factor 9G8. The interaction between mCBP and splicing factor 9G8 was confirmed in vivo. These results suggest a function of mCBP in RNA metabolism.

Animals↗

PubMed1996

The human hnRNP-M proteins: structure and relation with early heat shock-induced splicing arrest and chromosome mapping.

With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as early as 10 min after the onset of a heat shock at 45 degrees C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the nuclear matrix, the effect being reversed after a 6 h recovery at 37 degrees C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11 amino acid stretch from the originally published sequence. We also show that monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit in vitro splicing. Furthermore, stressing cells at 45 degrees C for 10 min is sufficient to abolish the splicing capacity of subsequently prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins are involved in splicing as well as in early stress-induced splicing arrest. Further in situ hybridization assays located the hnRNP-M encoding gene on human chromosome 19.

Amino Acid Sequence↗

PubMed1995

The gene encoding human splicing factor 9G8. Structure, chromosomal localization, and expression of alternatively processed transcripts.

The 9G8 factor is a 30-kDa member of the SR splicing factor family. We report here the isolation and characterization of the human 9G8 gene. This gene spans 7745 nucleotides and consists of 8 exons and 7 introns within the coding sequence, thus contrasting with the organization of the SC35/PR264 or RBP1 SR genes. We have located the human 9G8 gene in the p22-21 region of chromosome 2. The 5'-flanking region is GC-rich and contains basal promoter sequences and potential regulatory elements. Transfection experiments show that the 400-base pair flanking sequence has a promoter activity. Northern blot analysis of poly(A)+ RNA isolated from human fetal tissues has allowed us to identify five different species, generated by alternative splicing of intron 3, which may be retained or excised as a shorter version, as well as the use of two polyadenylation sites. We also show that the different isoforms are differentially expressed in the fetal tissues. The persistence of sequences between exon 3 and 4 results in the synthesis of a 9G8 protein lacking the SR domain which is expected to be inactive in constitutive splicing. Thus, our results raise the possibility that alternative splicing of intron 3 provides a mechanism for modulation of the 9G8 function.

Alternative Splicing↗

PubMed1995

Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing.

Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

Adenoviridae↗

PubMed1994

Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family.

By adopting a monoclonal antibody approach, we have identified a novel splicing factor of 35 kDa which we have termed 9G8. The isolation and characterization of cDNA clones indicate that 9G8 is a novel member of the serine/arginine (SR) splicing factor family because it includes an N-terminal RNA binding domain (RBD) and a C-terminal SR domain. The RNA binding domain of 9G8 is highly homologous to those of the SRp20 and RBP1 factors (79-71% identity), but the homology is less pronounced in the cases of SF2/ASF and SC35/PR264 (45-37% identity). Compared with the other SR splicing factors, 9G8 presents some specific sequence features because it contains an RRSRSXSX consensus sequence repeated six times in the SR domain, and a CCHC motif in its median region, similar to the zinc knuckle found in the SLU7 splicing factor in yeast. Complete immunodepletion of 9G8 from a nuclear extract, which is accompanied by a substantial depletion of other SR factors, results in a loss of splicing activity. We show that a recombinant 9G8 protein, expressed using a baculovirus vector and excluding other SR factors, rescues the splicing activity of a 9G8-depleted nuclear extract and an S100 cytoplasmic fraction. This indicates that 9G8 plays a crucial role in splicing, similar to that of the other SR splicing factors. This similarity was confirmed by the fact that purified human SC35 also rescues the 9G8-depleted extract. The identification of the 9G8 factor enlarges the essential family of SR splicing factors, whose members have also been proposed to play key roles in alternative splicing.

Amino Acid Sequence↗

PubMed1991

Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.

The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAs) by the use of three alternative 5'-splice sites. The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively. With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells. An in vitro analysis of the cis-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction. The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides. Complementation experiments show that the trans-acting factor(s) are micrococcal nuclease sensitive. They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection. We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells.

Adenoviridae↗

PubMed1991

Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns.

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

Adenovirus Early Proteins↗

PubMed1987

Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro.

We have developed an in vitro splicing system using a HeLa cell nuclear extract that is highly active for the alternative splicing of the natural E1A transcripts. The efficiency of using the three alternative 5' splice sites is strongly dependent on the ionic conditions in the reaction, and the simultaneous production of the 13S, 12S, and 9S mRNA species is observed only at appropriate salt concentrations. All the intermediate and final splicing products have been extensively characterized and it has been demonstrated that the same major branch site is used for all the alternative reactions. The ratio of 13S to 9S mRNAs formed is close to that observed in vivo early in infection, suggesting that most of the mechanisms giving rise to alternative splicing are preserved in vitro.

Adenovirus Early Proteins↗

PubMed1986

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.

Adenoviruses, Human↗