title: UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein.
abstract: Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5'-untranslated region (5'-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5'-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5'-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5'-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335-355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.
authors.affiliation: Department for Molecular Biomedical Research, VIB-Ghent University, Unit of Molecular Signal Transduction in Inflammation B-9052 Gent-Zwijnaarde, Belgium. Sigrid.Cornelis@dmbr.ugent.be
authors.forename: Sigrid
authors.initials: S
authors.lastname: Cornelis
authors.forename: Sandrine A
authors.initials: SA
authors.lastname: Tinton
authors.forename: Bert
authors.initials: B
authors.lastname: Schepens
authors.forename: Yanik
authors.initials: Y
authors.lastname: Bruynooghe
authors.forename: Rudi
authors.initials: R
authors.lastname: Beyaert
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D038941:Polypyrimidine Tract-Binding Protein / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
chemical_list: D020121:5' Untranslated Regions; C079950:CSDE1 protein, human; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D038941:Polypyrimidine Tract-Binding Protein