Uniquely Conserved Non-translated Regions are Involved in Generation of the Two Major Transcripts of Protein Phosphatase 2Cβ

• Published in: Journal of molecular biology 2001-09, Vol.312 (3), p.439-451
• Main Authors: Seroussi, Eyal, Shani, Nir, Ben-Meir, Daniella, Chajut, Ayelet, Divinski, Inna, Faier, Shlomit, Gery, Sigal, Karby, Shulamit, Kariv-Inbal, Zehavit, Sella, Osnat, Smorodinsky, Nechama I., Lavi, Sara
• Format: Article
• Language: English
• Subjects: alternative splicing; growth arrest; internal-ribosome-entry-site (IRES); phosphatase; phosphoprotein phosphatase 2C; PP2C
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.4967

Partial cDNAs of different isoforms of protein phosphatase 2Cβ (PP2Cβ or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cβ isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53kDa (PP2Cβl) and 43kDa (PP2Cβs) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cβ confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3′ exons that underlies the differences between them. The genomic structure of PP2Cβ is similar to that of other PP2C 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 variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5′-NTR is long (379bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cβ under CMV-promoter in 293 cells led to cell-growth arrest or cell death.