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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides
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Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

机译：鉴定选自随机寡核苷酸文库的两个短内部核糖体进入位点

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摘要

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reas- sayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expres- sion properties may be useful in the design of vectors for biotech- nology and gene therapy.
机译：控制mRNA翻译的序列可能在调节蛋白质水平中起关键作用。这样的元件之一是内部核糖体进入位点（IRES）。我们先前显示，编码Gtx同源域蛋白的mRNA的5'前导序列中的9-nt片段可以充当IRES。为了鉴定具有相似性质的其他短序列，我们设计了一种选择程序，该程序使用逆转录病毒载体表达双顺反子mRNA，分别编码增强的绿色和青色荧光蛋白作为第一和第二顺反子。第二顺反子的表达取决于顺反子间序列，并指示IRES活性。用两个逆转录病毒文库感染B104细胞，该文库在顺反子区域包含9或18 nt的随机序列。对表达两个顺反子的细胞进行分选，并在双萤光素酶双顺反子mRNA中重新确定从选定细胞中回收的序列的IRES活性。通过该程序鉴定出两个新颖的IRES，并且都包含与18S rRNA互补的区段。当任一部分的多个副本链接在一起时，IRES活动将大大增强。而且，这些合成的IRES在各种细胞类型中具有差异活性。这些性质类似于先前从Gtx mRNA鉴定的9-nt IRES模块的性质。这些结果提供了进一步的证据，即短核苷酸序列可以充当IRES，并支持某些细胞IRES可能由较短的功能模块组成的观点。识别具有特定表达特性的IRES模块的能力可能在设计用于生物技术和基因治疗的载体时很有用。

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