NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element
Abstract
The mRNA of human NF-κB 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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Footnotes
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↵5 Present address: University of Freiburg Medical School, ZKF Myocardial Metabolism, D-79106 Freiburg, Germany.
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Reprint requests to: M. Nourbakhsh, Institute of Pharmacology, Hannover Medical School OE5320, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany; e-mail: Nourbakhsh.Mahtab{at}MH-Hannover.de; Fax: +49 511 532 4081.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.545407.
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- Received March 7, 2007.
- Accepted May 3, 2007.
- Copyright © 2007 RNA Society