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Mariano A Garcia-Blanco

Publications and source records attributed to Mariano A Garcia-Blanco.

At least 19 recordsLinked to original sources
PubMed2006

Imaging the alternative silencing of FGFR2 exon IIIb in vivo.

Alternative splicing multiplies genomic coding capacity and regulates proteomic composition. A well-studied example of this plasticity leads to the synthesis of functionally distinct isoforms of the Fibroblast Growth Factor Receptor-2 (FGFR2). The regulation of this isoform diversity necessitates the silencing of FGFR2 exon IIIb, which is mediated by flanking intronic splicing silencers and the polypyrimidine tract binding protein (PTB). To visualize this splicing decision in vivo, we developed mice harboring a green fluorescent protein construct that reports on the silencing of exon IIIb. The animals also harbor a red fluorescent protein reporter of constitutive splicing as an allelic control. This dual reporter system revealed that in various organs and cell types the silencing of exon IIIb required the intronic silencers. In neurons, which do not express PTB, we observed robust silencer-dependent repression of exon IIIb, suggesting that the neural paralog, brain PTB, can take over this function. In the epidermis, however, the intronic silencers were not required for efficient silencing. This work provides a first glimpse at splicing regulation among different cell types in vivo and promises the drafting of an anatomic map of splicing decisions.

Alternative Splicing↗
PubMed2006

Assessing incomplete deprotection of microarray oligonucleotides in situ.

En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

Antibodies, Monoclonal↗
PubMed2006

Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity.

In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.

Alternative Splicing↗
PubMed2006

Alternative splicing: therapeutic target and tool.

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for therapy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense oligonucleotides, which can be employed to promote skipping of constitutive exons, inhibit inappropriately activated exons, or stimulate exons weakened by mutations. Additionally this manuscript evaluates methods that reprogram RNAs using reactions that recombine RNA molecules in trans. Preliminary, but exciting, results in these areas of investigation suggest that these methods could eventually lead to treatments in heretofore intractable ailments.

Alternative Splicing↗
PubMed2006

Human transcription elongation factor CA150 localizes to splicing factor-rich nuclear speckles and assembles transcription and splicing components into complexes through its amino and carboxyl regions.

The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.

Amino Acid Sequence↗
PubMed2006

Fox-2 mediates epithelial cell-specific fibroblast growth factor receptor 2 exon choice.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts occurs in a cell-type-specific manner leading to the mutually exclusive use of exon IIIb in epithelia or exon IIIc in mesenchyme. Epithelial cell-specific exon choice is dependent on (U)GCAUG elements, which have been shown to bind Fox protein family members. In this paper we show that FGFR2 exon choice is regulated by (U)GCAUG elements and Fox protein family members. Fox-2 isoforms are differentially expressed in IIIb+ cells in comparison to IIIc+ cells, and expression of Fox-1 or Fox-2 in the latter led to a striking alteration in FGFR2 splice choice from IIIc to IIIb. This switch was absolutely dependent on the (U)GCAUG elements present in the FGFR2 pre-mRNA and required critical residues in the C-terminal region of Fox-2. Interestingly, Fox-2 expression led to skipping of exon 6 among endogenous Fox-2 transcripts and formation of an inactive Fox-2 isoform, which suggests that Fox-2 can regulate its own activity. Moreover, the repression of exon IIIc in IIIb+ cells was abrogated by interfering RNA-mediated knockdown of Fox-2. We also show that Fox-2 is critical for the FGFR2(IIIb)-to-FGFR2(IIIc) switch observed in T Rex-293 cells grown to overconfluency. Overconfluent T Rex-293 cells show molecular and morphological changes consistent with a mesenchymal-to-epithelial transition. If overconfluent cells are depleted of Fox-2, the switch from IIIc to IIIb is abrogated. The data in this paper place Fox-2 among critical regulators of gene expression during mesenchymal-epithelial transitions and demonstrate that this action of Fox-2 is mediated by mechanisms distinct from those described for other cases of Fox activity.

Alternative Splicing↗
PubMed2005

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximately 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.

Alternative Splicing↗
PubMed2005

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb.

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.

Alternative Splicing↗
PubMed2005

In vitro coupled transcription splicing.

Many convincing studies published in recent years strongly support coupling of transcription and pre-mRNA processing. Despite key advances in our understanding of these processes, there is a lack of a robust in vitro system in which to study the mechanism of this coupling for complex pre-mRNAs. Here, we describe an in vitro system capable of transcribing and splicing complex transcripts with three and four exons. We also demonstrate how the system can be used to study exon silencing in vitro. We believe that this system will be a useful tool to study the mechanisms that mediate the coupling of transcription and pre-mRNA processing.

DNA-Directed RNA Polymerases↗
PubMed2005

Methods for the study of alternative splicing.

Alternative splicing multiplies the coding capacity of genes, which provides unparalleled complexity to the transcriptome and proteome. This increased complexity has clear repercussions for the regulation of gene expression in many organisms and for the balance between human health and disease. This significance, which has only recently been fully understood, has led to a growing number of studies that address the mechanisms that control alternative splicing decisions and the biological consequences of alternative RNA and protein isoforms. These studies have adapted or developed new methods and protocols that span computational biology, the biochemical analysis of in vitro reactions, model tissue culture systems, and in vivo studies. Many of these methods have been assembled in this volume to assist a newcomer to the study of alternative splicing and to expand the methodologies of laboratories already in the field.

Alternative Splicing↗
PubMed2005

The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation.

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.

DNA-Binding Proteins↗
PubMed2005

Making antisense of splicing.

Alternative splicing multiplies the coding capacity of the genome, resulting in an expanded proteome that provides many targets for therapy. In addition to creating this diverse pharmacoproteome, the process of splicing can be targeted by conventional and molecular therapies. Splicing as a therapeutic target is highlighted in this review, with a particular emphasis on oligonucleotide-based molecular approaches. These oligonucleotides can be used to promote skipping of constitutive exons, inhibit inappropriately activated exons, or stimulate exons weakened by mutations. Preliminary, but exciting, results suggest that these reagents could have clinical utility in treating previously intractable conditions.

Alternative Splicing↗
PubMed2004

MAZ elements alter transcription elongation and silencing of the fibroblast growth factor receptor 2 exon IIIb.

The fibroblast growth factor receptor 2 (FGFR2) gene exons IIIb and IIIc are alternatively spliced in a mutually exclusive and cell type-specific manner. FGFR2 exon choice depends on both activation and silencing. Exon IIIb silencing requires cis-acting elements upstream and downstream of the exon. To examine the influence of transcription on exon IIIb silencing, the putative RNA polymerase II (RNAPII)-pausing MAZ4 element was inserted at different positions within the FGFR2 minigene construct. MAZ4 insertions 5' to the upstream silencing elements or between exon IIIb and downstream silencing elements result in decreased silencing. An insertion 3' of the downstream silencing elements, however, has no effect on splicing. An RT-PCR elongation assay shows that the MAZ4 site in these constructs is likely to be a RNAPII pause site. Insertion of another RNAPII pause site into the minigene has a similar effect on exon IIIb silencing. Transfection of in vitro transcribed RNA demonstrates that the cell type specificity of FGFR2 alternative splicing requires co-transcriptional splicing. Additionally, changing the promoter alters both FGFR2 minigene splicing and the MAZ4 effect. We propose that RNAPII pauses at the MAZ4 elements resulting in a change in the transcription elongation complex that influences alternative splicing decisions downstream.

Alternative Splicing↗
PubMed2004

Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay.

Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. Our findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to NMD.

Alternative Splicing↗
PubMed2004

Alternative splicing in disease and therapy.

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting elements or trans-acting factors that lead to aberrant splicing and abnormal protein production. Correction of erroneous splicing is thus an important goal of molecular therapies. Recent experiments have used modified oligonucleotides to inhibit cryptic exons or to activate exons weakened by mutations, suggesting that these reagents could eventually lead to effective therapies.

Alternative Splicing↗
PubMed2004

Imaging alternative splicing in living cells.

We have developed an in vivo reporter of alternative splicing decisions that allows for the determination of FGF-R2 splicing patterns without the destruction of cells. This method has broad applications, including the study of other alternatively spliced genes in tissue culture and in whole animals, and may be useful in creating imaging markers for the study of tumor progression and metastasis. In this chapter, the authors present one example of this method using fluorescence reporters. As with any new assay, a series of experiments were performed to validate the method. This chapter documents some of these experiments.

Alternative Splicing↗
PubMed2003

Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing.

Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.

Animals↗