NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.
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.
Reboll
Marc René
MR
Institute of Pharmacology, Hannover Medical School, Hannover, Germany.
Oumard
André
A
Gazdag
Aniko Carla
AC
Renger
Isabelle
I
Ritter
Birgit
B
Schwarzer
Michael
M
Hauser
Hansjoerg
H
Wood
Monika
M
Yamada
Michiyuki
M
Resch
Klaus
K
Nourbakhsh
Mahtab
M
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
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