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Juan Valcárcel

Publications and source records attributed to Juan Valcárcel.

17 recordsLinked to original sources
PubMed2006

Fas-activated serine/threonine kinase (FAST K) synergizes with TIA-1/TIAR proteins to regulate Fas alternative splicing.

The factors and mechanisms that mediate the effects of intracellular signaling cascades on alternative pre-mRNA splicing are poorly understood. TIA-1 (T-cell intracellular antigen 1) and TIAR (TIA-1-related) proteins regulate alternative pre-mRNA splicing by promoting the use of suboptimal 5' splice sites followed by uridine-rich intronic enhancer sequences. These proteins promote, for example, inclusion of Fas receptor exon 6, which leads to an mRNA encoding a pro-apoptotic form of the receptor at the expense of the form that skips exon 6, which encodes an anti-apoptotic form. Fas-activated serine/threonine kinase (FAST K) is known to interact with and phosphorylate TIA-1. Here we have tested the possibility that FAST K influences alternative pre-mRNA splicing by affecting the activity of TIA-1/TIAR. Depletion of FAST K form Jurkat cells leads to skipping of exon 6 from endogenous Fas transcripts. Conversely, FAST K overexpression enhances exon 6 inclusion of Fas reporters transfected in HeLa cells. Consistent with the possibility that the effects of FAST K are mediated by changes in the function of TIA-1/TIAR, the effects of FAST K overexpression (i) are largely suppressed by depletion of TIA-1 and TIAR and (ii) are significantly compromised by mutation of a TIA-1/TIAR-responsive enhancer present downstream of exon 6 5' splice site. Furthermore, in vitro phosphorylation of TIA-1 by FAST K results in enhanced U1 snRNP recruitment. Interestingly, this enhancement is not due to increased binding of TIA-1 to the pre-mRNA. Taken together, the results connect Fas signaling with the activity of splicing factors that modulate Fas alternative splicing, suggesting the existence of an autoregulatory loop that could serve to amplify Fas responses.

Alternative Splicing↗
PubMed2006

Unweaving the meanings of messenger RNA sequences.

In addition to protein-coding information, mRNAs harbor regulatory sequences necessary for appropriate processing of their precursors. Goren et al. (2006) and Wang et al. (2006) explore the diversity of these signals and the rules by which they function.

Alternative Splicing↗
PubMed2006

Intron removal requires proofreading of U2AF/3' splice site recognition by DEK.

Discrimination between splice sites and similar, nonsplice sequences is essential for correct intron removal and messenger RNA formation in eukaryotes. The 65- and 35-kD subunits of the splicing factor U2AF, U2AF65 and U2AF35, recognize, respectively, the pyrimidine-rich tract and the conserved terminal AG present at metazoan 3' splice sites. We report that DEK, a chromatin- and RNA-associated protein mutated or overexpressed in certain cancers, enforces 3' splice site discrimination by U2AF. DEK phosphorylated at serines 19 and 32 associates with U2AF35, facilitates the U2AF35-AG interaction and prevents binding of U2AF65 to pyrimidine tracts not followed by AG. DEK and its phosphorylation are required for intron removal, but not for splicing complex assembly, which indicates that proofreading of early 3' splice site recognition influences catalytic activation of the spliceosome.

Adenosine Triphosphate↗
PubMed2006

The expanding transcriptome: the genome as the 'Book of Sand'.

The central dogma of molecular biology inspired by classical work in prokaryotic organisms accounts for only part of the genetic agenda of complex eukaryotes. First, post-transcriptional events lead to the generation of multiple mRNAs, proteins and functions from a single primary transcript, revealing regulatory networks distinct in mechanism and biological function from those controlling RNA transcription. Second, a variety of populous families of small RNAs (small nuclear RNAs, small nucleolar RNAs, microRNAs, siRNAs and shRNAs) assemble on ribonucleoprotein complexes and regulate virtually all aspects of the gene expression pathway, with profound biological consequences. Third, high-throughput methods of genomic analysis reveal that RNAs other than non-protein-coding RNAs (ncRNAs) represent a major component of the transcriptome that may perform novel functions in gene regulation and beyond. Post-transcriptional regulation, small RNAs and ncRNAs provide an expanding picture of the transcriptome that enriches our views of what genes are, how they operate, evolve and are regulated.

Animals↗
PubMed2005

Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition.

Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.

Alternative Splicing↗
PubMed2005

Building specificity with nonspecific RNA-binding proteins.

Specificity is key to biological regulation. Two families of RNA binding proteins, heterogeneous nuclear ribonucleoproteins and serine-arginine-rich proteins, were initially thought to have redundant or nonspecific biochemical functions. Recently, members of these families have been found as components of distinct regulatory complexes with highly specific and essential roles in mRNA metabolism. Here we discuss the basis for their functional specificity and the mechanisms of action of some of their characteristic protein domains.

Amino Acids↗
PubMed2004

Alternative splicing microarrays reveal functional expression of neuron-specific regulators in Hodgkin lymphoma cells.

Alternative splicing provides a versatile mechanism of gene regulation, which is often subverted in disease. We have used customized oligonucleotide microarrays to interrogate simultaneously the levels of expression of splicing factors and the patterns of alternative splicing of genes involved in tumor progression. Analysis of RNAs isolated from cell lines derived from Hodgkin lymphoma tumors indicate that the relative abundance of alternatively spliced isoforms correlates with transformation and tumor grade. Changes in expression of regulators were also detected, and a subset sample was confirmed at the protein level. Ectopic expression of neuron-specific splicing regulatory proteins of the Nova family was observed in some cell lines and tumor samples, correlating with expression of a neuron-specific mRNA isoform of JNK2 kinase. This microarray design can help assess the role of alternative splicing in a variety of biological and medical problems and potentially serve as a diagnostic tool.

Alternative Splicing↗
PubMed2004

Diversity of vertebrate splicing factor U2AF35: identification of alternatively spliced U2AF1 mRNAS.

U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.

Alternative Splicing↗
PubMed2003

U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5' splice sites.

The splicing factor U2AF(65), U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF(65) can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF(65) is critical for U1 recruitment, and we discuss the role of its RNA-RNA annealing activity in this novel function of U2AF(65).

Cell Nucleus↗
PubMed2003

Getting to the heart of a splicing enhancer.

Dinucleotide CA repeats of variable length in an intron of the human endothelial nitric oxide synthase gene act as regulatory signals for splicing of the gene's pre-mRNA. Repeat length-dependent binding of hnRNP L to the repeats provides an intriguing link between splicing efficiency, nitric oxide synthesis and cardiovascular disease.

Dinucleotide Repeats↗
PubMed2002

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites.

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5' splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5' ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5' ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5' ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator.

Alternative Splicing↗
PubMed2002

Biochemical function of female-lethal (2)D/Wilms' tumor suppressor-1-associated proteins in alternative pre-mRNA splicing.

Genetic and molecular data have implicated the Drosophila gene female-lethal (2)d (fl (2)d) in alternative splicing regulation of genes involved in sexual determination. Sex-specific splicing is under the control of the female-specific regulatory protein sex-lethal (SXL). Co-immunoprecipitation and mass spectrometry results indicate that SXL and FL (2)D form a complex and that the protein VIRILIZER and a Ran-binding protein implicated in protein nuclear import are also present in complexes containing FL (2)D. A human homolog of FL (2)D was identified and cloned. Interestingly, this gene encodes a protein (WTAP) that was previously found to interact with the Wilms' tumor suppressor-1 (WT1), an isoform of which binds to and co-localizes with splicing factors. Alternative splicing of transformer pre-mRNA, a target of SXL regulation, was affected by immunodepletion of hFL (2)D/WTAP from HeLa nuclear extracts, thus arguing for a biochemical function of FL (2)D/WTAP proteins in splicing regulation.

Alternative Splicing↗
PubMed2002

Optimization of oligonucleotide-based DNA microarrays.

Oligonucleotide-based DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms. Here we report a systematic study of the sensitivity, specificity and dynamic range of microarray signals and their dependence on the labeling and hybridization conditions as well as on the length, concentration, attachment moiety and purity of the oligonucleotides. Both a controlled set of in vitro synthesized transcripts and RNAs from biological samples were used in these experiments. An algorithm is presented that allows the efficient selection of oligonucleotides able to discriminate a single nucleotide mismatch. Critical parameters for various applications are discussed based on statistical analysis of the results. These data will facilitate the design and standardization of custom-made microarrays applicable to gene expression profiling and sequencing analyses.

Animals↗
PubMed2002

Splicing regulation at the second catalytic step by Sex-lethal involves 3' splice site recognition by SPF45.

The Drosophila protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. Here we show that U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with SXL blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' ss generated by a mutation that causes human beta-thalassemia.

Adenine Nucleotides↗
PubMed2002

Regulation of alternative pre-mRNA splicing.

The first wave of bioinformatic studies that followed genome and complementary DNA sequencing projects revealed that alternative splicing of messenger RNA precursors (pre-mRNAs) contributes substantially to transcriptome complexity in higher eukaryotes. Together with the realisation of the impact of the process on cell differentiation, development and disease, these studies portray alternative splicing as a fundamental component of gene regulation. Both detailed mechanistic studies and genome-wide analyses will be necessary to unravel the molecular basis for cell type-specific splice site selection. This paper will highlight some recent progress and future challenges for functional genomics and bioinformatics in this rapidly developing area.

Alternative Splicing↗
PubMed2001

Alternative splicing and genome complexity.

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. It has recently been proposed as a mechanism by which higher-order diversity is generated. Here we show, using large-scale expressed sequence tag (EST) analysis, that among seven different eukaryotes the amount of alternative splicing is comparable, with no large differences between humans and other animals.

Alternative Splicing↗