Biomedical subjects

Andrew P Baraniak

Publications and source records attributed to Andrew P Baraniak.

6 recordsLinked to original sources

PubMed2006

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

Alternative Splicing↗

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

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↗

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↗

PubMed2003

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.

Alternative Splicing↗

PubMed2003

Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay.

The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing.

Alternative Splicing↗