NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element
NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element
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DOI：
10.1261/rna.545407
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发表时间：
2007-08-01
期刊：
影响因子：
4.5
通讯作者：
Nourbakhsh, Mahtab
中科院分区：
文献类型：
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作者：
Reboll, Marc Rene;Oumard, Andre;Nourbakhsh, Mahtab
人NF-κ B抑制因子（NRF）的mRNA含有长的5 '非翻译区（UTR），其通过帽非依赖性机制将核糖体引导至下游起始密码子。人和小鼠NRF mRNA的核苷酸（nt）序列的比较揭示了在起始密码子附近的150 nt片段的高度同一性。在这里，我们表明，该地区构成了一个最小的内部核糖体进入段（IRES）模块。酶促RNA结构分析揭示了NRF IRES模块的二级结构模型。该模块的点突变分析确定了短的14-nt RNA元件（nt 640-653）作为IRES功能的介体。IRES结合细胞蛋白的纯化和随后的ESI/MS/MS序列分析导致RNA结合蛋白JKTBP 1的鉴定。EMSA实验表明，JKTBP 1在NRF IRES模块中的14-nt RNA元件（nt 579-639）的上游结合。JKTBP 1的过表达显著增强双顺反子构建体中NRF IRES模块的活性。此外，siRNA实验证明内源性JKTBP 1的下调降低了NRF IRES活性和内源性NRF蛋白的水平。本研究的数据表明，JKTBP 1和14-nt元件独立地介导NRF IRES活性。
The mRNA of human NF-kappa B 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.