Splicing mediates the activity of four putative cellular internal ribosome entry sites
Splicing mediates the activity of four putative cellular internal ribosome entry sites
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DOI：
10.1073/pnas.0710650105
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发表时间：
2008-03-25
影响因子：
11.1
通讯作者：
Logg, Christopher R.
中科院分区：
文献类型：
--
作者：
Baranick, Brian T.;Lemp, Nathan A.;Logg, Christopher R.
越来越多的细胞mrna被认为具有内部核糖体进入位点（IRESs），这些序列允许转录本独立于其5'端和帽结构进行翻译。虽然双双性分析是检测IRES活性序列的标准方法，但如果使用的双双性结构产生了意想不到的单反性rna，则可能产生假阳性结果。利用双反子报告系统和绿色荧光蛋白标记的逆转录病毒来评估先前报道的6种细胞IRES，我们发现其中4种含有3'剪接位点，其活性是IRES明显功能所必需的，并通过剪接形成单反子转录本。生物信息学分析显示，在三个假定的IRESs中鉴定的3'剪接位点用于其原生mrna中，而第四个可能是在cDNA克隆过程中产生的人工序列。我们的研究结果表明，需要通过仔细的RNA结构分析来重新检查其他已报道的细胞IRES，以排除剪接作为感知IRES活性的来源。
A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.