Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2C beta

Partial cDNAs of different isoforms of protein phosphatase 2C beta (PP2C be ta or PPM1 beta) have been characterized in mammals. We disclose here the f ull cDNAs of two major PP2C beta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2C beta1) and 43 kDa (P P2C betas) polypeptides, respectively. The isoforms are co-expressed ubiqui tously with the highest level in skeletal muscle, as assessed by Northern-b lot analysis. Western and in situ analyses using monoclonal antibodies agai nst PP2C beta confirmed the existence of two isoforms in the cytoplasm. Com parative sequence analysis revealed that both cDNAs consist of six exons wi th an alternate usage of the 3 ' exons that underlies the differences betwe en them. The genomic structure of PP2C beta is similar to that of other PP2 C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both var iants of the ending exon include large non-coding regions. All nontranslate d regions (NTRs) are highly conserved between the orthologous genes, indica ting their regulatory function. The 5 ' -NTR is long (379 bp), includes ups tream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. In troduction of this NTR element into a bi-luciferase expression-cassette ena bled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpre ssion of PP2C beta under CMV-promoter in 293 cells led to cell-growth arres t or cell death. (C) 2001 Academic Press.