Biomedical subjects

J Valcárcel

Publications and source records attributed to J Valcárcel.

32 records · Page 2Linked to original sources

PubMed1997

Targeting of U2AF65 to sites of active splicing in the nucleus.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

Amino Acid Sequence↗

PubMed1997

Novel functions for 'nuclear factors' in the cytoplasm: the Sex-lethal paradigm.

In recent years, novel functions for a number of nuclear factors have been uncovered in the cytoplasm, mainly at the level of translation. These factors behave as multifunctional regulators of gene expression and many play key roles in cell differentiation and development. One of the best characterized examples is that of Sex-lethal (SXL), an RNA-binding protein that is expressed in female Drosophila flies and controls sex determination and dosage compensation. Recent findings indicate that SXL, a paradigmatic regulator of splicing, also controls translation of target mRNAs. This review attempts to summarize this evidence and provide an overview of 'nuclear factors' with roles in translation.Copyright 1998 Academic Press Limited

Journal Article↗

PubMed1996

Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected].

The mammalian splicing factor U2AF65 binds to the polypyrimidine tract adjacent to the 3' splice site and promotes assembly of U2 small nuclear ribonucleoprotein on the upstream branch point, an interaction that involves base pairing with U2 small nuclear RNA (snRNA). U2AF65 contains an RNA binding domain, required for interaction with the polypyrimidine tract, and an arginine-serine-rich (RS) region, required for U2 snRNP recruitment and splicing. Here it is reported that binding of U2AF65 to the polypyrimidine tract directed the RS domain to contact the branch point and promoted U2 snRNA-branch point base pairing even in the absence of other splicing factors. Analysis of RS domain mutants indicated that the ability of U2AF65 to contact the branch point, to promote the U2 snRNA-branch point interaction, and to support splicing are related activities, requiring only a few basic amino acids. Thus, the U2AF65 RS domain plays a direct role in modulating spliceosomal RNA-RNA interactions.

Amino Acid Sequence↗

PubMed1996

The SR protein family: pleiotropic functions in pre-mRNA splicing.

A family of proteins with arginine-serine-rich domains has recently come into the limelight of studies on the mechanisms of constitutive and regulated pre-mRNA splicing. Implicated in an ever increasing variety of functions, these proteins act as driving forces during spliceosome assembly and also play decisive roles in alternative splice-site selection, suggesting that they are crucial players in the regulation of splicing during cell differentiation and development.

Animals↗

PubMed1995

Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.

In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. Here it is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site.

Animals↗

PubMed1995

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.

Base Sequence↗

PubMed1993

The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA.

Somatic sexual differentiation in Drosophila melanogaster involves a cascade of regulated splicing events and provides an attractive model system for the analysis of alternative splicing mechanisms. The protein Sex-lethal (Sxl) activates a female-specific 3' splice site in the first intron of transformer (tra) pre-mRNA while repressing an alternative non-sex-specific site. We have developed an in vitro system that recapitulates this regulation in a manner consistent with genetic, transfection and fly transformation studies. Using this system, we have determined the molecular basis of the splice site switch. Here we show that Sxl inhibits splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A splicing 'effector' domain present in U2AF but absent from Sxl accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to Sxl converts it from a splicing repressor to an activator and renders it unable to mediate splice-site switching.

Alternative Splicing↗

PubMed1993

Splicing of influenza virus matrix protein mRNA expressed from a simian virus 40 recombinant.

Influenza virus RNA segment 7 encodes two proteins, M1 and M2, depending on the optional removal of an intron from its primary transcript. To investigate the mechanism of this regulated splicing, an influenza virus segment 7 cDNA was cloned under the control of simian 40 virus (SV40) early promoter and poly(A) signals in an SV40 recombinant virus (SVM), and expressed in COS-1 cells. Expression of both M1 and M2 proteins was detected in SVM-infected cells, suggesting (i) the appropriate splicing events to generate M2 mRNA occur in these cells and (ii) significant amounts of unspliced M1 mRNA are transported to the cytoplasm. Analysis of the relative proportion of M2 mRNA to mRNA3 indicated that the use of the alternative 5' splice sites is reversed in SVM-infected cells compared with those infected with influenza virus. In addition, a different intranuclear distribution of segment 7 transcripts was found in each type of infected cell. We speculate that these differences in splicing efficiency and splice site choice might be related to different subnuclear localizations of segment 7 transcripts synthesized by the different transcriptional machineries.

Alternative Splicing↗

PubMed1992

Nuclear transport of influenza virus polymerase PA protein.

The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein.

Animals↗

PubMed1992

Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff.

The kinetics of cellular mRNA decay in influenza virus-infected cells have been studied by means of blot hybridization using as probes cloned cDNAs of alpha- and beta-actin, alpha- and beta-tubulin and vimentin. Both cellular mRNAs isolated from the cytoplasmic fractions as well as total cell mRNAs showed a rapid decay, with up to 50% concentration reductions at infection times at which influenza virus M1 mRNA was still not detectable. In contrast, these cellular mRNAs were stable in uninfected cells. To ascertain the possible role of mRNA degradation in the cellular protein synthesis shutoff, the kinetics of protein synthesis in infected cells were examined by two-dimensional gel electrophoresis of extracts pulse-labelled at several times after viral infection. The synthesis of the cellular proteins was reduced, showing kinetics paralleling those of mRNA decay. It is proposed that influenza virus infection induces the destabilization of mRNAs and that this mRNA degradation is, at least in part, responsible for cellular protein synthesis shutoff.

Actins↗

PubMed1992

Heterogeneity of the mutation rates of influenza A viruses: isolation of mutator mutants.

The rates of mutation to the mar (monoclonal antibody-resistant) genotype of individual influenza virus plaque isolates, obtained from a stock generated after two successive cloning steps, have been determined by the fluctuation test. When a random sample of 60 clones was analyzed, 7 contained a proportion of mar mutants significantly higher than the average, and among them, 2 showed a mutation rate two to three times higher than the average value obtained for the virus population when the hemagglutinin-specific monoclonal antibody 2G10 was used. In order to look for mutants with higher mutation rates, a systematic search was carried out with a nonmutagenized virus stock, and several clones with increased mutation rates were isolated. One of them (mut43) was characterized further and was shown to have a mutation rate three to four times higher than that of the virus population at the sites defined by two nonoverlapping, hemagglutinin-specific monoclonal antibodies as well as at the site defined by a neuraminidase-specific monoclonal antibody. These results indicate that the mutation rate of an influenza virus is a weighted average of the contributions of a heterogeneous population. The consequences of this fact for the adaptive evolution of influenza viruses are discussed.

Antibodies, Monoclonal↗

PubMed1991

Regulated M1 mRNA splicing in influenza virus-infected cells.

Influenza virus RNA segment 7 generates three poly(A)+ RNAs, M1 mRNA, M2 mRNA and mRNA3, the last of which has almost no coding capacity; M2 mRNA and mRNA3 derive from M1 mRNA by removal of a single intron. The kinetics of M1 and M2 mRNA accumulation in the cytoplasm of productively infected cells were studied by means of a quantitative RNA protection assay; the ratio of M2 mRNA to M1 mRNA increased 2.7-fold during the course of infection. To analyse the basis for this change, the kinetics of M1 and M2 mRNA synthesis and nuclear accumulation, their stability and nucleocytoplasmic transport were studied. Under the experimental conditions used, the synthesis of segment 7-specific RNA showed a peak at 4 h post-infection and continued later at a slower rate. The half-lives of M1 and M2 mRNAs were indistinguishable (2.73 h for M1 mRNA and 2.70 h for M2 mRNA) and the kinetics of nucleocytoplasmic transport in vivo or in vitro showed no preference for either mRNA early or late in infection. Consequently, regulation at the level of mRNA splicing is proposed. Using the mRNA synthesis and stability data, a simulation was performed to predict the change in splicing efficiency required to account for the mRNA accumulation results. The best fit was obtained when splicing efficiency changed about 20 times during a period in which viral gene expression was maximal.

Animals↗

PubMed1989

Permanent cell lines that show temperature-dependent expression of adenovirus virus-associated RNA.

Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection.

Acetyltransferases↗

PubMed1989

Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.

Antibodies, Monoclonal↗