Biomedical subjects

Stéphan Vagner

Publications and source records attributed to Stéphan Vagner.

7 recordsLinked to original sources

PubMed2006

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries.

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.

3' Untranslated Regions↗

PubMed2004

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA.

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.

5' Untranslated Regions↗

PubMed2004

Pharmacological-based translational induction of transgene expression in mammalian cells.

In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level. To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor. This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras. This membrane-delocalized translation factor is inactive unless liberated in the cytosol. Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space. Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.

Alkyl and Aryl Transferases↗

PubMed2003

A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons.

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.

Alternative Splicing↗

PubMed2003

IRESdb: the Internal Ribosome Entry Site database.

Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon. These sequences are very diverse and are present in a growing list of mRNAs. Novel IRES sequences continue to be added to public databases every year and the list of unknown IRESes is certainly still very large. The IRES database is a comprehensive WWW resource for internal ribosome entry sites and presents currently available general information as well as detailed data for each IRES. It is a searchable, periodically updated collection of IRES RNA sequences. Sequences are presented in FASTA form and hotlinked to NCBI GenBank files. Several subsets of data are classified according to the viral taxon (for viral IRESes), to the gene product function (for cellular IRESes), to the possible cellular regulation or to the trans-acting factor that mediates IRES function. This database is accessible at http://ifr31w3.toulouse.inserm.fr/IRESdatabase/.

Animals↗

PubMed2002

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing.

Splicing and 3'-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3'-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3'-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3'-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3'-end processing, which contributes to 3'-terminal exon definition.

Cell Nucleus↗

PubMedDate not supplied

Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons.

The use of several translation initiation codons in a single mRNA, by expressing several proteins from a single gene, contributes to the generation of protein diversity. A small, yet growing, number of mammalian mRNAs initiate translation from a non-AUG codon, in addition to initiating at a downstream in-frame AUG codon. Translation initiation on such mRNAs results in the synthesis of proteins harbouring different amino terminal domains potentially conferring on these isoforms distinct functions. Use of non-AUG codons appears to be governed by several features, including the sequence context and the secondary structure surrounding the codon. Selection of the downstream initiation codon can occur by leaky scanning of the 43S ribosomal subunit, internal entry of ribosome or ribosomal shunting. The biological significance of non-AUG alternative initiation is demonstrated by the different subcellular localisations and/or distinct biological functions of the isoforms translated from the single mRNA as illustrated by the two main angiogenic factor genes encoding the fibroblast growth factor 2 (FGF2) and the vascular endothelial growth factor (VEGF). Consequently, the regulation of alternative initiation of translation might have a crucial role for the biological function of the gene product.

Animals↗