Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing

Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Ричмонд, Virginia, United States
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/2003; 1625(2):153-64. DOI: 10.1016/S0167-4781(02)00601-2
Source: PubMed


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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    • "LocusID 24241 maps to probe sets 1369116_a_at, 1369117_at and 1370775_a_at (Table 4), and corresponds to the rat calcitonin alpha/calcitonin gene-related peptide (CGRP) gene. The mammalian Calca/CGRP gene has six exons, and is considered a model gene for the study of alternative splicing [27]. Splicing together the first four exons produces the mRNA for calcitonin alpha, whereas splicing together exons one to three, five and six produces the mRNA for CGRP (Figure 6). "
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    • "The DNA to mRNA transcription with alternative RNA splicing was found in 1982 for Calcitonin/CGRP gene expression [2]. We draw focus to model Calcitonin/CGRP gene expression while combining other recent biological knowledge [7] [36]. As in Figure 2, the Calcitonin gene expression consists of four introns and five exons, and the transcription process progresses with step1, step2, step3 and step4. "
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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.
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