Molecular structure and alternative splicing of the human carboxypeptidase M gene.
Using RACE technology the 5' and 3' ends of human carboxypeptidase M (CPM) mRNA were determined and found to be divergent from the published sequence. With these results the complete structure of the human CPM gene was established based on the human genome sequence in the GenBank database. The gene was shown to contain 9 exons comprising at least 75 kb of genomic sequence. A novel first exon of 30 bp was identified and an upstream promoter sequence containing several transcription factor binding sites was found by computer analysis. Furthermore, the ATG starting codon was detected defining an open reading frame of 1329 bp that codes for a protein of 443 residues. Additionally, the polyadenylation site was discovered, determining a 3' noncoding region of 2000 nucleotides. The exon-intron boundaries diverged substantially compared to those of the other basic carboxypeptidases, CPD, CPE, CPN, and AEBP1. Cloning and sequencing of RT-PCR products from different tissues revealed alternative splicing of exons 3 and 5, which results in the generation of four different mRNA isoforms. RNA extracted from tumor tissues contained more CPM mRNA than control tissue, suggesting an upregulation of CPM expression in tumors and raising the question of the role of this enzyme in cancer.
Pessoa
Luciana G
LG
Department of Biophysics, Universidade Federal de São Paulo, Brazil.
da Silva
Ismael D Guerreiro
ID
Baptista
Heloisa A
HA
Pesquero
Jorge L
JL
Paiva
Antonio C M
AC
Bader
Michael
M
Pesquero
João B
JB
D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D058851:GPI-Linked Proteins; D015723:Gene Library; D006801:Humans; D007438:Introns / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D022821:RNA Splice Sites / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017421:Sequence Analysis; D024363:Transcription Initiation Site; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D058851:GPI-Linked Proteins; D022821:RNA Splice Sites; C058479:carboxypeptidase M; D008666:Metalloendopeptidases