Volume 16, Issue 1, Pages 107-116 (October 2004) AlkB Restores the Biological Function of mRNA and tRNA Inactivated by Chemical Methylation  Rune Ougland, Chun-Mei Zhang, Aivar Liiv, Rune F. Johansen, Erling Seeberg, Ya-Ming Hou, Jaanus Remme, Pål Ø. Falnes  Molecular Cell  Volume 16, Issue 1, Pages 107-116 (October 2004) DOI: 10.1016/j.molcel.2004.09.002

Figure 1 Effect of AlkB-Mediated Repair on the Translation of Chemically Methylated mRNA (A and B) mRNALuc was treated with the indicated concentrations of MMS, and then incubated in the absence (closed symbols) or presence (open symbols) of 0.83 μM AlkB, followed by translation in a reticulocyte lysate containing [35S]methionine. (A) The translation products were separated by SDS-PAGE and visualized by phosphorimagery. (B) The amount of luciferase was measured by phosphoimager quantitation of the gel bands (circles) and by measuring the luciferase activity in the translation mixture (triangles). The amount of translation product has been expressed as percent of the level obtained in the case of nonmethylated mRNALuc. (C and D) mRNALuc was treated with 100 mM MMS and then incubated in the presence of various amounts of AlkB or hABH3, followed by translation in a reticulocyte lysate in the presence of [35S]methionine. (C) The translation products were separated by SDS-PAGE and visualized by phosphorimagery. (D) The amount of luciferase was measured by phosphoimager quantitation of the gel bands (circles) and by measuring the luciferase activity (triangles) in the translation mixture. Closed symbols, AlkB. Open symbols, hABH3. The amount of luciferase has been expressed as percent of the level obtained in the presence of 0.83 μM AlkB. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 2 Increased Aminoacylation of Chemically Methylated tRNAPhe upon Treatment with AlkB or hABH3 (A and B) E. coli total tRNA was treated with different concentrations of DMS under native (A) or semidenaturing (B) conditions, followed by incubation in the absence (open circles) or presence (closed circles) of 6.7 μM AlkB. The ability of tRNAPhe to function as acceptor for aminoacylation was then measured in vitro in the presence of E. coli S-100 proteins and [14C]phenylalanine. The level of aminoacylation has been expressed as percent of that obtained with tRNAPhe not treated with DMS. Error bars indicate the standard deviation of triplicate samples. (C) tRNAPhe was treated with 100 mM DMS under native conditions and then incubated in the presence of various amounts of AlkB (closed circles) or hABH3 (open circles) as indicated. Subsequently, the ability of tRNAPhe to function as acceptor for aminoacylation was measured. The aminoacylation is expressed as relative increase compared to the level obtained without repair enzyme. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 3 Repair by AlkB or hABH3 Increases the Ability of Chemically Methylated tRNAPhe to Support Poly(U)-Directed Translation (A–C) Similar experiments as those described in the corresponding panels in Figure 2, except that the concentration of AlkB in (A) and (B) was 11 μM, and instead of measuring the aminoacylation of tRNAPhe, the ability of tRNAPhe to support protein synthesis was measured in an in vitro system containing poly(U), E. coli S-100 proteins, and ribosomes. (D) tRNAPhe was treated with 100 mM DMS under native conditions and then incubated in the absence or presence of AlkB (11 μM), the cofactors 2-oxoglutarate and ferrous iron (Fe/2-Ox), or combinations thereof, as indicated. Finally, the ability of the tRNAPhe to support poly(U)-directed translation was measured. Error bars represent the range between duplicate samples (A and B), or the standard deviation of triplicate samples (D). Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 4 AlkB-Mediated Removal of Chemically Introduced Methyl Lesions from E. coli tRNA E. coli tRNA (10 μg) was incubated in the absence or presence of 300 mM DMS and then enzymatically degraded to nucleosides, which were subsequently analyzed by reverse-phase HPLC. Asterisks indicate peaks that were present both in the DMS-treated and untreated tRNA and are thus likely to represent minor modified bases occurring naturally in tRNA. (A) DMS-treated tRNA. (B) Magnification of part of the chromatogram shown in (A). (C) DMS-treated tRNA subsequently treated with 5.5 μM AlkB. (D) Untreated tRNA. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 5 Activity of Human and Bacterial AlkB Proteins on [14C]methylated tRNA [14C]methylated tRNA was incubated with proteins as indicated for 30 min at 37°C. Subsequently, the tRNA was precipitated by ethanol precipitation and the radioactivity in the supernatant measured by scintillation counting. Error bars represent the standard deviation of triplicate samples. Closed circles, AlkB; open circles, hABH2; triangles, hABH3. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 6 Mapping of Positions in tRNAPhe Repaired by AlkB (A) Sequence and cloverleaf structure of E. coli tRNAPhe, where the sites of complementarity to primer 1 and primer 2 are indicated by “*”. The major sites of AlkB repair are shown by arrows. (B) Primer extension by primer 1 of control tRNA (lane 1), AlkB-repaired tRNA (lane 2), DMS-modified tRNA (lane 3), and AlkB-repaired, DMS-modified tRNA (lane 4). The DMS-modified sites are indicated by “|” (lane 3) and those repaired by AlkB are indicated by closed circles (lane 4). (C) Primer extension by primer 2 of control tRNA (lane 1), AlkB-repaired tRNA (lane 2), DMS-modified tRNA (lane 3), and AlkB-repaired of the DMS-modified tRNA (lane 4). The DMS-modified sites are indicated by “|” (lane 3) and those repaired by AlkB are indicated by closed circles (lane 4). Size markers for primer extension are shown as “M.” (D) The ratios of extension block versus readthough at each of the sites of repair in (B) and (C) were determined by measuring the band intensity before and after the AlkB repair (lane 3 versus lane 4). The ratios have been calculated from three different experiments, and error bars represent the standard deviation. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)

Figure 7 Repair of 1-meA in E. coli tRNA In Vivo The E. coli strains AB 1157 (wild-type), HK 82 (alkB mutant), or HK82/AlkB (AlkB overexpressed) were metabolically labeled with [3H]adenine, then treated with DMS and incubated for varying time periods in DMS-free medium. tRNA was then isolated and degraded to nucleosides, which were subjected to reverse-phase HPLC for determination of the 1-meA content. (A) The bacteria were exposed to different concentrations of DMS, and tRNA was isolated directly after the DMS treatment. (B) The bacteria were exposed to 1 mM DMS and then incubated for the indicated time periods before tRNA was isolated. Error bars represent the standard deviation of triplicate samples. Similar results were obtained in three independent experiments. Molecular Cell 2004 16, 107-116DOI: (10.1016/j.molcel.2004.09.002)