The effect of RNA base lesions on mRNA translation

Abstract

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.

Figure 1.

Structures of RNA lesions investigated: 8-oxo-7,8-dihydroadenosine (8-oxo-rA), 8-oxo-7,8-dihydroguanosine (8-oxo-rG), 5-hydroxycytidine (5-HO-rC), 5-hydroxyuridine (5-HO-rU), 1,N⁶-ethenoadenosine (ε-rA), 3,N⁴-ethenocytidine (ε-rC) and abasic site (rAS); rib = ribose.

Figure 2.

(A) Synthesis of the puromycin mRNA constructs via splint ligation of synthetic oligonucleotides. X is the position where the lesions were introduced and Y indicates the encoded amino acids, 9 = triethylene glycol linker, P = puromycin. (B) Translation assay: the hybrid template is labeled with a radioactive phosphate and used in a standard rabbit reticulocyte translation assay. The results are analyzed by PAGE.

Figure 3.

Fusion-translation product of an mRNA construct bearing a rA at position X (X = A). T = template; F = fusion-translation mixture; a) = template; b) = fusion product.

Figure 4.

Fusion-translation product obtained from the mRNA construct containing X = rC or X = 5-HO-rC. Column 1: rC; column 2: 5-HO-rC (5-O-protected); columns 3–5: 5-HO-rC (5-O-deprotected) using differently prepared mRNA constructs; column 3: 5-O-deprotected before translation; column 4: 5-O-deprotected before splint ligation; column 5: 5-O-deprotected before the splint ligation without subsequent heating. Lane F: fusion-translation assay products; lane T: mRNA construct as control. Arrows: a) = template; b) = truncated fusion product; and c) = full-length fusion product.

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.

Figure 6.

Fusion-translation products of templates bearing a natural base (rA, rC, rG, rU) or a lesion corresponding to the position X. T = mRNA construct; F = fusion translation. Arrows: a) = template; b) = truncated fusion products; and c) = full-length fusion products.