The effect of RNA base lesions on mRNA translation

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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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)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Küpfer, Pascal and 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:

12 Sep 2017 07:20

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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