Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.
Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.
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DOI：
10.1093/nar/gks530
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发表时间：
2012-09
影响因子：
14.9
通讯作者：
Mauro VP
中科院分区：
文献类型：
--
作者：
Burman LG;Mauro VP
高等真核生物核糖体RNA（rRNA）的加工、组装和功能的分析由于缺乏使rRNA能够被修饰然后功能性地检查的表达系统而受到阻碍。考虑到这种系统的潜在用途，我们已经开发了一种用于哺乳动物18S rRNA的系统。我们将序列标签插入小鼠18S rRNA的扩增片段3中，以通过杂交监测表达和切割。鉴定了赋予对帕塔霉素抗性的突变，通过选择性阻断内源性（帕塔霉素敏感性）亚基的翻译，允许对含有合成18S rRNA的40S核糖体亚基进行功能分析。rRNA构建体在转染的细胞中适当表达，显示正确加工，掺入约15%的40S亚基，并且基于各种标准正常发挥功能。经过严格的分析，该系统被用来调查的重要性，侧翼序列的前体转录18S rRNA。虽然缺失分析支持U3 snoRNA需要结合位点，但它表明不需要位于18S rRNA侧翼的5′外转录间隔区的大片段和整个第一内转录间隔区。这种方法的成功开启了核糖体功能分析的可能性，并在基础研究和合成生物学中得到应用。
Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5′ external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.
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影响因子：
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