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NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.
Reboll, Marc René Oumard, André Gazdag, Aniko Carla Renger, Isabelle Ritter, Birgit Schwarzer, Michael Hauser, Hansjoerg Wood, Monika Yamada, Michiyuki Resch, Klaus
Reboll, Marc René
Oumard, André
Gazdag, Aniko Carla
Renger, Isabelle
Ritter, Birgit
Schwarzer, Michael
Hauser, Hansjoerg
Wood, Monika
Yamada, Michiyuki
Resch, Klaus
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2007-08
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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.
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NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element. 2007, 13 (8):1328-40 RNA
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en
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1355-8382