An internal ribosome entry site mediates translation of lymphoid enhancer factor-1.
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'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-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'-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'-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'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-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.
Jimenez
Judith
J
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697-4025, USA.
Jang
Gwendolyn M
GM
Semler
Bert L
BL
Waterman
Marian L
ML
D020121:5' Untranslated Regions / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003593:Cytoplasm / Q000502:physiology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004285:Dogs; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012315:RNA Caps / Q000502:physiology; D012333:RNA, Messenger / Q000502:physiology; D012270:Ribosomes / Q000502:physiology*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D012315:RNA Caps; D012333:RNA, Messenger; D014157:Transcription Factors