Expression of RUNX2 isoforms: involvement of cap-dependent and cap-independent mechanisms of translation

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.     

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.     

Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs

Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.     

Connexin43 mRNA contains a functional internal ribosome entry site

A reporter gene construct was used to study the regulation of connexin43 (Cx43) expression, the major gap junction protein found in heart and uterus, in transfected cell lines. The construct had the firefly luciferase gene under the control of the Cx43 promoter. Inclusion of the 5'-untranslated region (UTR) of the mRNA in the construct increased luciferase expression by 70%. A bicistronic vector assay demonstrated that the Cx43 5'-UTR contains a strong internal ribosome entry site (IRES). Deletion analysis localized the IRES element to the upstream portion of the 5'-UTR.     

Internal ribosome entry site mediates protein synthesis in yeast Pichia pastoris

The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.