Calabretta, Alessandro; Küpfer, Pascal A.; Leumann, Christian J. (2015). The effect of RNA base lesions on mRNA translation. Nucleic acids research, 43(9), pp. 4713-4720. Information Retrieval Ltd. 10.1093/nar/gkv377
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Nucl. Acids Res.-2015-Calabretta-4713-20-1.pdf - Published Version Available under License Creative Commons: Attribution (CC-BY). Download (6MB) | Preview |
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 32P-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 35S-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.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
UniBE Contributor: |
Küpfer, Pascal, Leumann, Christian |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
ISSN: |
0305-1048 |
Publisher: |
Information Retrieval Ltd. |
Language: |
English |
Submitter: |
Christian Leumann |
Date Deposited: |
20 May 2015 15:08 |
Last Modified: |
05 Dec 2022 14:47 |
Publisher DOI: |
10.1093/nar/gkv377 |
PubMed ID: |
25897124 |
Additional Information: |
Date: May 19, 2015 |
BORIS DOI: |
10.7892/boris.69086 |
URI: |
https://boris.unibe.ch/id/eprint/69086 |
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