Inhibition of 6-methyladenine formation decreases the translation efficiency of dihydrofolate reductase transcripts.
Cycloleucine was used to inhibit the formation of internal N6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells cultured in cycloleucine produced transcripts deficient in N6-methyladenosine residues and the 2'-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2'-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2'-O-methylation in the cap. These experiments therefore suggest that an inhibition of N6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.
Tuck
M T
MT
Department of Chemistry and Biochemistry, Ohio University, Athens 45701, USA. mtuck1@ohiou.edu
Wiehl
P E
PE
Pan
T
T
D000225:Adenine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D000818:Animals; D003515:Cycloleucine / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis; D009705:Nucleosides / Q000032:analysis; D014176:Protein Biosynthesis / Q000187:drug effects*; D012315:RNA Caps / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012260:Ribonucleases; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000225:Adenine; D003515:Cycloleucine; D009363:Neoplasm Proteins; D009705:Nucleosides; D012315:RNA Caps; D012260:Ribonucleases; D013762:Tetrahydrofolate Dehydrogenase; C000730567:6-methyladenine