An internal ribosome entry site mediates translation of lymphoid enhancer factor-1

Abstract

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5 0 -untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5 0 -UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator b-catenin. Truncated LEF-1 lacks the b-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5 0 -UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5 0 -UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5 0 -UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5 0 -UTR shows that maximal IRES activity requires the majority of the 5 0 -UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.

PITX2 and β-Catenin Interactions Regulate Lef-1 Isoform Expression

“…The Lef-1 FL isoform is produced from the Lef-1 P1 promoter, and the N-terminally truncated Lef-1 ⌬N113 isoform is synthesized from the Lef-1 P2 promoter in intron 2 ( Fig. 4A) (30,34). The 55-and 57-kDa Lef-1 products are produced from the same P1 promoter and may arise from alternative splicing or posttranslational modifications (30,34,45).…”

“…4A) (30,34). The 55-and 57-kDa Lef-1 products are produced from the same P1 promoter and may arise from alternative splicing or posttranslational modifications (30,34,45). However, CHO cells endogenously express only the 39-kDa isoform (Fig.…”

“…The fulllength Lef-1 transcript contains a large 5Ј untranslated region synthesized using a separate P1 promoter and utilizes a capindependent mechanism for translation of the full-length Lef-1 containing the ␤BD (30,34). This Lef-1 activity is countered by the expression of a truncated Lef-1 protein that lacks the ␤BD and can compete for binding to Wnt target genes.…”

“…This Lef-1 activity is countered by the expression of a truncated Lef-1 protein that lacks the ␤BD and can compete for binding to Wnt target genes. This shorter Lef-1 transcript (Lef-1 ⌬N113) could act as an inhibitory isoform, due to its ability to interact with cofactors that also bind Lef-1 FL, as well as compete for DNA binding sites, and thus has been termed growth suppressing (27,30,34). However, both Lef-1 isoforms can stimulate cell differentiation.…”

“…However, both Lef-1 isoforms can stimulate cell differentiation. Interestingly, in colon cancer cells, only the LEF-1 FL transcript is produced, and the loss of balance between the long and short forms may affect cancer progression (30,34). Therefore, it is essential to understand the molecular functions of these two Lef-1 isoforms.…”