title: NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.
abstract: The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Hannover, Germany.
authors.forename: Marc René
authors.initials: MR
authors.lastname: Reboll
authors.forename: André
authors.initials: A
authors.lastname: Oumard
authors.forename: Aniko Carla
authors.initials: AC
authors.lastname: Gazdag
authors.forename: Isabelle
authors.initials: I
authors.lastname: Renger
authors.forename: Birgit
authors.initials: B
authors.lastname: Ritter
authors.forename: Michael
authors.initials: M
authors.lastname: Schwarzer
authors.forename: Hansjoerg
authors.initials: H
authors.lastname: Hauser
authors.forename: Monika
authors.initials: M
authors.lastname: Wood
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Yamada
authors.forename: Klaus
authors.initials: K
authors.lastname: Resch
authors.forename: Mahtab
authors.initials: M
authors.lastname: Nourbakhsh
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020033:Protein Isoforms / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism*; D012270:Ribosomes
chemical_list: D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C111986:HNRPDL protein, human; C484734:Hnrpdl protein, mouse; C111027:NKRF protein, human; C522199:NKRF protein, mouse; D020033:Protein Isoforms; D012097:Repressor Proteins; D012261:Ribonucleoproteins