Calabretta Alessandro, Küpfer Pascal A, Leumann Christian J
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland
Nucleic Acids Res. 2015 May 19;43(9):4713-20. doi: 10.1093/nar/gkv377. Epub 2015 Apr 20.
The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell. Here we report on the effect of the commonly known RNA base-lesions 8-oxo-rG, 8-oxo-rA, ε-rC, ε-rA, 5-HO-rC, 5-HO-rU and the RNA abasic site (rAS) on ribosomal translation. To this end we have developed an in vitro translation assay based on the mRNA display methodology. A short synthetic mRNA construct containing the base lesion in a predefined position of the open reading frame was (32)P-labeled at the 5'-end and equipped with a puromycin unit at the 3'-end. Upon in vitro translation in rabbit reticulocyte lysates, the encoded peptide chain is transferred to the puromycin unit and the products analyzed by gel electrophoresis. Alternatively, the unlabeled mRNA construct was used and incubated with (35)S-methionine to prove peptide elongation of the message. We find that all base-lesions interfere substantially with ribosomal translation. We identified two classes, the first containing modifications at the base coding edge (ε-rC, ε-rA and rAS) which completely abolish peptide synthesis at the site of modification, and the second consisting of 8-oxo-rG, 8-oxo-rA, 5-HO-rC and 5-HO-rU that significantly retard full-length peptide synthesis, leading to some abortive peptides at the site of modification.
与氧化损伤的DNA不同，氧化损伤的RNA的生物学效应很少被研究，尽管它对任何活细胞都构成威胁。在此，我们报告了常见的RNA碱基损伤8-氧代-rG、8-氧代-rA、ε-rC、ε-rA、5-羟基-rC、5-羟基-rU以及RNA无碱基位点（rAS）对核糖体翻译的影响。为此，我们基于mRNA展示方法开发了一种体外翻译测定法。一个在开放阅读框的预定义位置包含碱基损伤的短合成mRNA构建体在5'-末端进行了（32）P标记，并在3'-末端配备了嘌呤霉素单元。在兔网织红细胞裂解物中进行体外翻译时，编码的肽链转移到嘌呤霉素单元，并通过凝胶电泳分析产物。或者，使用未标记的mRNA构建体并与（35）S-甲硫氨酸一起孵育，以证明该信息的肽链延伸。我们发现所有碱基损伤都对核糖体翻译有实质性干扰。我们确定了两类，第一类包含碱基编码边缘的修饰（ε-rC、ε-rA和rAS），这些修饰在修饰位点完全消除肽合成，第二类由8-氧代-rG、8-氧代-rA、5-羟基-rC和5-羟基-rU组成，它们显著延迟全长肽合成，在修饰位点导致一些流产肽。
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