SYNTHESIS OF CIRCULAR RNA IN BACTERIA AND YEAST USING RNA CYCLASE RIBOZYMES DERIVED FROM A GROUP-I INTRON OF PHAGE-T4
SYNTHESIS OF CIRCULAR RNA IN BACTERIA AND YEAST USING RNA CYCLASE RIBOZYMES DERIVED FROM A GROUP-I INTRON OF PHAGE-T4
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DOI：
10.1073/pnas.91.8.3117
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
ARES, M
中科院分区：
文献类型：
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作者：
FORD, E;ARES, M
由于环状RNA难以表达，限制了对环状RNA功能和RNA拓扑结构的研究。为了克服这一点，将来自噬菌体T4 td基因的I组内含子在外周环（L6a）中分裂并重排，使得3'半内含子和3'剪接位点在单个外显子的上游，5'剪接位点和5'半内含子在单个外显子的下游。I组剪接反应将内部外显子RNA切割成环状（RNA环化酶核酶活性）。我们表明，外源序列可以放置在外显子和环状体外。在大肠杆菌和酵母中表达这样的构建体（RNA环化酶核酶）导致环状RNA在这些生物体中积累。在酵母中，RNA环化酶核酶可以从受调控的启动子如mRNA表达，其含有5'前导区和3'尾随区以及核前mRNA内含子。RNA环化酶核酶在RNA结构和功能的问题上有广泛的应用，包括RNA转运或功能的末端要求、RNA拓扑学、反义或核酶基因控制元件的功效以及极长多肽的生物合成。
Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.