Abstract
The gene encoding the murine calcitonin receptor (mCTR) was isolated, and the exon/intron structure was determined. Analysis of transcripts revealed novel cDNA sequences, new alternative exon splicing in the 5'-untranslated region, and three putative promoters (P1, P2, and P3). The longest transcription unit is greater than 67 kilobase pairs, and the location of introns within the coding region of the mCTR gene (exons E3-E14) are identical to those of the porcine and human CTR genes. We have identified novel cDNA sequences that form three new exons as well as others that add 512 base pairs to the 5' side of the previously published cDNA, thereby extending exon E1 to 682 base pairs. Two of these novel exons are upstream of exon E2 and form a tripartite exon E2 (E2a, E2b, and E2c) in which E2a is utilized by promoter P2 with variable splicing of E2b. The third new exon (E3b') lies between E3a and E3b and is utilized by promoter P3. Analysis of mCTR mRNAs has revealed that the three alternative promoters give rise to at least seven mCTR isoforms in the 5' region of the gene and generate 5'-untranslated regions of very different lengths. Analysis by reverse transcription-polymerase chain reaction shows that promoters P1 and P2 are utilized in osteoclasts, brain, and kidney, whereas promoter P3 appears to be osteoclast-specific. Using transiently transfected reporter constructs, promoter P2 has activity in both a murine kidney cell line (MDCT209) and a chicken osteoclast-like cell line (HD-11EM), whereas promoter P3 is active only in the osteoclast-like cell line. These transfection data confirm the osteoclast specificity of promoter P3 and provide the first evidence that the CTR gene is regulated in a tissue-specific manner by alternative promoter utilization.
Highlights
¶ Present address: Dept. of Conservative Dentistry, Faculty of Dentistry, Prince of Songkla University, Hatyai, Songkla, Thailand 90112
To search for the existence of splicing isoforms of the 5Ј-end of the murine calcitonin receptor (mCTR) gene, additional 5Ј-Rapid Amplification of cDNA Ends (5Ј-RACE) was performed in which the first PCR step employed primers AP1 and E6-R
This report establishes that the mCTR gene is transcribed from three different promoters in a tissue-specific fashion and that these three primary transcripts are spliced within the 5Ј-UTR to generate seven mRNA isoforms
Summary
Some of the human [23] and porcine CTR (pCTR) [22] genomic sequences have been cloned, little is known about the mechanism of transcriptional regulation for the CTR gene in osteoclasts or in other tissues in which it is expressed. Analysis of mCTR mRNAs reveals that the transcripts from the three promoters are spliced to yield seven different 5Ј-UTR structures Analysis by both RT-PCR and transient transfection of promoter-luciferase reporter constructs shows that the P1 promoter (located upstream of an expanded exon E1) and the P2 promoter (located upstream of exon E2a) are utilized in osteoclasts, brain, and kidney, whereas the P3 promoter (located upstream of the novel exon E3bЈ) appears to be exclusively utilized in osteoclasts. The P2 promoter of mCTR is highly homologous to the promoter region previously defined for pCTR in kidney cells These studies provide the first evidence that CTR is regulated in a tissuespecific manner by alternative promoter utilization and that there is a unique promoter (P3) that regulates CTR expression only in osteoclasts
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