Figure 1. Identification and validation of PTENα.

(A) Sequence of the 5′-UTR region of Homo sapiens PTEN mRNA. Two potential CUG sites (red and pink) as well as the normal ATG start site (green) are highlighted.

(B) The 5′-UTR of PTEN containing the 16-bp CUG-centered palindromic motif is evolutionary conserved. Phylogenetic analysis of the 5′-UTR of PTEN mRNA in bonobo (Pan paniscus), wild boar (Sus scrofa), horse (Equus caballus), cattle (Bos taurus), killer whale (Orcinus orca) and mouse (Mus musculus). The 16-bp palindromic sequence is highlighted in a green box and the ATG start codon of canonical PTEN is in a blue box.

(C) PTEN cds with a CUG⁵¹³-starting 5′-UTR region (PTENα) was constructed under a CMV promoter and expressed with and without a N-terminal FLAG tag. Human HEK293T cells were transfected with indicated expression plasmids, followed by Western blotting analysis using a PTEN monoclonal antibody against its C-terminal region.

(D) A different set of constructs of PTEN and PTENα with a C-terminal GFP tag, in which one of the two possible CTG sites (CTG⁵¹³ or CTG⁶³⁹) was mutated to CTA or GGA.

(E-G) Mutation of CTG⁵¹³ but not CTG⁶³⁹ eliminates the expression of PTENα. C-terminal GFP-tagged PTENα expression plasmids with and without CTG⁵¹³>CTA or CTG⁵¹³>GGA mutation(s) were introduced in human HCT116 colon cancer cells, followed by detection of GFP-PTENα variants. E, The expression of GFP-tagged PTEN or PTENα was confirmed by Western blotting using a GFP antibody. F and G, mutation of CTG⁵¹³ but not CTG⁶³⁹ into CTA or GGA results in the disappearance of PTENα. PTENα-G and PTEN-G, PTENα or PTEN with a C-terminal GFP tag.

See also Figure S1.