Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.
ABSTRACT The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.
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ABSTRACT: 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.Molecular and Cellular Biology 03/2007; 27(3):830-41. · 5.37 Impact Factor
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ABSTRACT: The research on modeling and simulation of complex biological systems is getting more important in Systems Biology. In this respect, we have developed Hybrid Function Petri net (HFPN) that was newly developed from existing Petri net because of their intuitive graphical representation and their capabilities for mathematical analyses. However, in the process of modeling metabolic, gene regulatory or signal transduction pathways with the architecture, we have realized three extensions of HFPN, (i) an entity should be extended to contain more than one value, (ii) an entity should be extended to handle other primitive types, e.g. boolean, string, (iii) an entity should be extended to handle more advanced type called object that consists of variables and methods, are necessary for modeling biological systems with Petri net based architecture. To deal with it, we define a new enhanced Petri net called hybrid functional Petri net with extension (HFPNe). To demonstrate the effectiveness of the enhancements, we model and simulate with HFPNe four biological processes that are diffcult to represent with the previous architecture HFPN.Genome informatics. International Conference on Genome Informatics 02/2004; 15(1):180-97.
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ABSTRACT: The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. Sequences in the human calcitonin-specific fourth exon function as an exonic splice enhancer (ESE) which is required for incorporation of exon 4 into calcitonin mRNA. Deletion of these sequences from the rat calcitonin/CGRP gene was reported to have no effect on calcitonin splicing. We demonstrate that sequences in the rat calcitonin/CGRP fourth exon act as an ESE. In addition, we observed that three proteins in HeLa nuclear extract, of apparent molecular weights of 40, 55 and 85 kDa, specifically interact with the exon 4 ESE. The 40-kDa protein is human transformer 2β (hTra2β), a homolog of the Drosophila splice regulator transformer 2. hTra2β is required for calcitonin splicing in vitro, one of the first biological functions identified for hTra2β. The 55-kDa protein is SRp55, a member of the SR family of phosphoproteins. Binding of SRp55 to an ESE required for calcitonin mRNA splicing suggests that the different levels of SRp55 present in different cell types may regulate calcitonin/CGRP alternative splicing.Biochimica Et Biophysica Acta-gene Structure and Expression - BBA-GENE STRUCT EXPRESS. 01/2003; 1625(2):141-152.