Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties.
Pax-8, a member of the paired box-containing gene family, was shown to be coexpressed with Pax-2 in several human kidney carcinoma cell lines. Four different Pax-8 mRNA isoforms, a to d, were cloned from one of these cell lines by polymerase chain reaction amplification, and the Pax-8 gene was isolated from a human cosmid library. Analysis of the exon-intron structure of Pax-8 revealed that the four mRNA isoforms arise by alternative splicing, resulting in inclusion or exclusion of exon 7 and/or exon 8 sequences. All four Pax-8 proteins retain the paired domain as their DNA-binding motif and recognize DNA in the same manner as do the closely related Pax-2 and BSAP (Pax-5) proteins. The Pax-8a and Pax-8b isoforms end in a serine/threonine/tyrosine-rich sequence, while the C terminus of Pax-8c and Pax-8d is translated in a different, proline-rich reading frame. Transient transfection experiments revealed that Pax-8 isoforms a and b, but not c and d, strongly stimulate transcription from a promoter containing six copies of a paired-domain recognition sequence. The same four mRNA variants were also detected by RNase protection analysis in the mouse embryo and adult kidney, thus indicating evolutionary conservation of Pax-8 mRNA splicing. A different splice pattern was observed in the developing placenta, which expresses two new variants, Pax-8e and Pax-8f, instead of transcripts b to d. Expression of these mRNAs is high at embryonic day 9.5 and is gradually reduced until Pax-8a is the predominant transcript in the 12.5-day placenta. In the embryo, however, the synthesis of mRNAs b to d is initially low and then increases relative to that of Pax-8a. Hence, alternative splicing of Pax-8 gene transcripts not only generates six different Pax-8 variants but is also temporally and spatially regulated during early mouse development.
Kozmik
Z
Z
Research Institute of Molecular Pathology, Vienna, Austria.
Kurzbauer
R
R
Dörfler
P
P
Busslinger
M
M
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004330:Drosophila; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009687:Nuclear Proteins; D051762:PAX2 Transcription Factor; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051762:PAX2 Transcription Factor; C497318:PAX2 protein, human; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; C497319:Pax2 protein, mouse; C499496:Pax8 protein, mouse; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA