Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties.

Abstract

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 Par-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 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 ofPax-8 gene transcripts not only generates six different Pax-8 variants but is also temporally and spatially regulated during early mouse development.

Pax8 has a key role in thyroid cell differentiation

“…The four Pax8 variants encode for proteins that differ in the C terminus and thus could differ, as suggested in previous studies (15,25), in transactivation properties. In all our experiments, we have used the full-length Pax8 cDNA, namely the Pax8a isoform.…”

“…Four Pax8 spliced transcripts have been described: Pax8a, Pax8b, Pax8c, and Pax8d (15,25). Pax8a, the most relevant isoform, contains all of the 10 exons and encodes for the most abundant Pax8 species.…”

Zebrafish Pax9 Encodes Two Proteins with Distinct C-terminal Transactivating Domains of Different Potency Negatively Regulated by Adjacent N-terminal Sequences

“…Alternative splicing has also been reported for other Pax genes. Similar to zebrafish Pax9, human PAX2 and PAX8 give rise to proteins differing in their Cterminal sequences (69,70). For human PAX6, alternative splicing leads to the generation of two proteins differing by a 14-amino acid insertion in the paired domain that alters the DNA binding-specificity (63).…”

Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival

“…This pattern is maintained in the mature organism. However, in a few adult tissues, PAX genes are expressed in a restricted fashion (Kozmik et al, 1993;St-Onge et al, 1997;Eccles, 1998). In contrast, abnormal cell growth and proliferation is associated with high levels of PAX gene expression in several diseases occurring in otherwise terminally differentiated tissues (Bernasconi et al, 1996;Winyard et al, 1996;Murer et al, 2002).…”