Alternative RNA processing--its role in regulating expression of calcitonin/calcitonin gene-related peptide

Different 3' coding exons in the rat calcitonin gene are used to generate distinct mRNAs encoding either the hormone calcitonin in thyroidal C-cells or a new neuropeptide referred to as calcitonin gene-related peptide in neuronal tissue, indicating the RNA processing regulation is one strategy used in tissue-specific regulation of gene expression in the brain. Although the two mRNAs use the same transcriptional initiation site and have identical 5' terminal sequences, their 3' termini are distinct. The polyadenylation sites for calcitonin and calcitonin gene-related peptide mRNAs are located at the end of the exons 4 and 6, respectively. Termination of transcription after the calcitonin exon does not dictate the production of calcitonin mRNA, because transcription proceeds through both calcitonin and calcitonin gene-related peptide exons irrespective of which mRNA is ultimately produced. In isolated nuclei, both polyadenylation sites appear to be utilized; however, the proximal (calcitonin) site is preferentially used in nuclei from tissues producing calcitonin mRNA. These data suggest that the mechanism dictating production of each mRNA involves the selective use of alternative polyadenylation sites.

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Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.5999-6011.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 5999-6011

Author(s):

J M Yeakley ◽

F Hedjran ◽

J P Morfin ◽

N Merillat ◽

M G Rosenfeld ◽

...

Keyword(s):

Alternative Splicing ◽

Calcitonin Gene Related Peptide ◽

Regulatory Sequences ◽

Primary Transcript ◽

Tissue Specific ◽

Related Peptide ◽

Thyroid C Cells ◽

Calcitonin Gene ◽

Constitutive Splicing ◽

Specific Regulation

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

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Calcitonin/calcitonin gene-related peptide transcription unit: tissue-specific expression involves selective use of alternative polyadenylation sites.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2151 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2151-2160 ◽

Cited By ~ 106

Author(s):

S G Amara ◽

R M Evans ◽

M G Rosenfeld

Keyword(s):

Regulation Of Gene Expression ◽

Alternative Polyadenylation ◽

Calcitonin Gene Related Peptide ◽

Transcription Unit ◽

Specific Expression ◽

Tissue Specific ◽

Related Peptide ◽

Calcitonin Gene ◽

Polyadenylation Sites ◽

Specific Regulation

Different 3' coding exons in the rat calcitonin gene are used to generate distinct mRNAs encoding either the hormone calcitonin in thyroidal C-cells or a new neuropeptide referred to as calcitonin gene-related peptide in neuronal tissue, indicating the RNA processing regulation is one strategy used in tissue-specific regulation of gene expression in the brain. Although the two mRNAs use the same transcriptional initiation site and have identical 5' terminal sequences, their 3' termini are distinct. The polyadenylation sites for calcitonin and calcitonin gene-related peptide mRNAs are located at the end of the exons 4 and 6, respectively. Termination of transcription after the calcitonin exon does not dictate the production of calcitonin mRNA, because transcription proceeds through both calcitonin and calcitonin gene-related peptide exons irrespective of which mRNA is ultimately produced. In isolated nuclei, both polyadenylation sites appear to be utilized; however, the proximal (calcitonin) site is preferentially used in nuclei from tissues producing calcitonin mRNA. These data suggest that the mechanism dictating production of each mRNA involves the selective use of alternative polyadenylation sites.

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Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.5999 ◽

1993 ◽

Vol 13 (10) ◽

pp. 5999-6011 ◽

Cited By ~ 32

Author(s):

J M Yeakley ◽

F Hedjran ◽

J P Morfin ◽

N Merillat ◽

M G Rosenfeld ◽

...

Keyword(s):

Alternative Splicing ◽

Calcitonin Gene Related Peptide ◽

Regulatory Sequences ◽

Primary Transcript ◽

Tissue Specific ◽

Related Peptide ◽

Thyroid C Cells ◽

Calcitonin Gene ◽

Constitutive Splicing ◽

Specific Regulation

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

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Tissue-Specific Alternative RNA Processing in Calcitonin/ Calcitonin Gene-Related Peptide Gene Expression

Cellular Physiology and Biochemistry ◽

10.1159/000154683 ◽

1993 ◽

Vol 3 (3-4) ◽

pp. 181-196 ◽

Cited By ~ 3

Author(s):

Ronald B. Emeson ◽

Joanne M. Yeakley

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Calcitonin Gene Related Peptide ◽

Tissue Specific ◽

Related Peptide ◽

Calcitonin Gene ◽

Specific Alternative ◽

Peptide Gene

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An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7135 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7135-7142 ◽

Cited By ~ 41

Author(s):

H Lou ◽

Y Yang ◽

G J Cote ◽

S M Berget ◽

R F Gagel

Keyword(s):

Splice Site ◽

Rna Processing ◽

Mammalian Species ◽

Calcitonin Gene Related Peptide ◽

Regulatory Sequences ◽

Human Calcitonin ◽

Terminal Exon ◽

Related Peptide ◽

Calcitonin Gene ◽

Splice Site Sequence

Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.

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