Characterization of the human and mouse genes for the alpha subunit of type II prolyl 4-hydroxylase. Identification of a previously unknown alternatively spliced exon and its expression in various tissues.
Prolyl 4-hydroxylase (4-PH) catalyzes the formation of 4-hydroxyproline in -X-Pro-Gly- sequences and has a central role in the synthesis of all collagens. We report here on the cloning and characterization of the genes encoding the catalytic alpha(II) subunits of the human and mouse type II 4-PH [alpha(II)]2beta2 tetramers. The human and mouse genes are approximately 34.6 kb and 30.3 kb in size, respectively, and both consist of 16 exons. The translation initiation codons are located in exon 2, and the sizes of the exons consisting entirely of coding sequences are conserved in the two genes, varying from 54 to 240 bp, whereas the exons 1, containing the transcription initiation sites and 5' untranslated sequences, are 546 bp and 293 bp in the human and mouse, respectively. The sizes of the introns vary from 48 to 49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but they and introns 1 contain several motifs that may act as transcription factor binding sites, including those for Sox9, which regulates chondrocyte-specific expression of collagens II, IX and XI. Unlike the human alpha(I) gene, the alpha(II) genes do not contain an alternatively spliced exon homologous to exon 9. However, a novel mutually exclusively spliced alternative exon 12a was identified in both genes. The nucleotide and amino-acid sequence identities between the 60-bp exon 12a and 66-bp exon 12b are about 35% and 45%, respectively, in both human and mouse genes. PCR analyses showed that both types of exon 12 are expressed in all tissues studied, except for adult leukocytes that expressed only mRNAs containing exon 12b sequences. Insect cell expression studies showed that a recombinant alpha(II) subunit containing amino acids coded by exon 12a associated with the beta subunit to form a fully active enzyme tetramer.
Nokelainen
M
M
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry, University of Oulu, Finland.
Nissi
R
R
Kukkola
L
L
Helaakoski
T
T
Myllyharju
J
J
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D024363:Transcription Initiation Site
D021122:Protein Subunits; D022821:RNA Splice Sites; D012333:RNA, Messenger; D011393:Procollagen-Proline Dioxygenase