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The effect of RNA base lesions on mRNA translation.

Calabretta A, Küpfer PA, Leumann CJ - Nucleic Acids Res. (2015)

Bottom Line: The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.

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Fusion-translation experiment of Figure 4 using 35S-Met and non-labeled mRNA construct (left, lanes 1–5) or 32P-labeled template and non-labeled Met (right, lanes 1′–5′). Samples 1–5 are as in Figure 4; B corresponds to a fusion-translation experiment with 35S-Met without mRNA construct. Arrows: a) = template; b) = fusion product; and c) = truncated fusion products.
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Figure 5: Fusion-translation experiment of Figure 4 using 35S-Met and non-labeled mRNA construct (left, lanes 1–5) or 32P-labeled template and non-labeled Met (right, lanes 1′–5′). Samples 1–5 are as in Figure 4; B corresponds to a fusion-translation experiment with 35S-Met without mRNA construct. Arrows: a) = template; b) = fusion product; and c) = truncated fusion products.

Mentions: To further confirm the presence of the peptide in the fusion-translation products, the same experiment was repeated with a non-labeled mRNA construct and 35S-Met in the translation mixture. The corresponding fusion translation product that was then compared with the same product using the 32P-labeled construct (Figure 5). The 32P-labeled products were repeatedly diluted until the bands showed comparable intensity in the gel. As expected in the absence of the mRNA construct, no band arising from the fusion product is visible despite the presence of 35S-methionine in the mixture (Figure 5, lane B). However, for the case of X = rC, and the O-5-protected 5-HO-rC clear bands are visible (lanes 1 and 2) that have the same electrophoretic mobility as the corresponding bands in lanes 1′ and 2′, arising from the 32P-labeled mRNA construct. When using the unprotected 5-HO-rC lesion (lane 3) two bands are detectable representing the full-length and the aborted fusion translation products. Lanes 4 and 5, finally, show only very faint bands, in agreement with a lower efficiency of translation. The corresponding experiments with the 32P-labeled mRNA construct gave comparable bands, including, of course, those of the non-translated mRNA construct that shows the highest mobility (lanes 3′–5′). Thus this experiment unambiguously confirms that mRNA–peptide conjugates are formed during in vitro translation and that truncation is associated with incomplete peptide synthesis.


The effect of RNA base lesions on mRNA translation.

Calabretta A, Küpfer PA, Leumann CJ - Nucleic Acids Res. (2015)

Fusion-translation experiment of Figure 4 using 35S-Met and non-labeled mRNA construct (left, lanes 1–5) or 32P-labeled template and non-labeled Met (right, lanes 1′–5′). Samples 1–5 are as in Figure 4; B corresponds to a fusion-translation experiment with 35S-Met without mRNA construct. Arrows: a) = template; b) = fusion product; and c) = truncated fusion products.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482091&req=5

Figure 5: Fusion-translation experiment of Figure 4 using 35S-Met and non-labeled mRNA construct (left, lanes 1–5) or 32P-labeled template and non-labeled Met (right, lanes 1′–5′). Samples 1–5 are as in Figure 4; B corresponds to a fusion-translation experiment with 35S-Met without mRNA construct. Arrows: a) = template; b) = fusion product; and c) = truncated fusion products.
Mentions: To further confirm the presence of the peptide in the fusion-translation products, the same experiment was repeated with a non-labeled mRNA construct and 35S-Met in the translation mixture. The corresponding fusion translation product that was then compared with the same product using the 32P-labeled construct (Figure 5). The 32P-labeled products were repeatedly diluted until the bands showed comparable intensity in the gel. As expected in the absence of the mRNA construct, no band arising from the fusion product is visible despite the presence of 35S-methionine in the mixture (Figure 5, lane B). However, for the case of X = rC, and the O-5-protected 5-HO-rC clear bands are visible (lanes 1 and 2) that have the same electrophoretic mobility as the corresponding bands in lanes 1′ and 2′, arising from the 32P-labeled mRNA construct. When using the unprotected 5-HO-rC lesion (lane 3) two bands are detectable representing the full-length and the aborted fusion translation products. Lanes 4 and 5, finally, show only very faint bands, in agreement with a lower efficiency of translation. The corresponding experiments with the 32P-labeled mRNA construct gave comparable bands, including, of course, those of the non-translated mRNA construct that shows the highest mobility (lanes 3′–5′). Thus this experiment unambiguously confirms that mRNA–peptide conjugates are formed during in vitro translation and that truncation is associated with incomplete peptide synthesis.

Bottom Line: The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.

Show MeSH
Related in: MedlinePlus