Cell cycle-coupled variation in topoisomerase IIalpha mRNA is regulated by the 3'-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation.
Mammalian topoisomerase IIalpha (Topo II) is a highly regulated enzyme essential for many cellular processes including the G(2) cell cycle checkpoint. Because Topo II gene expression is regulated posttranscriptionally during the cell cycle, we investigated the possible role of the 3'-untranslated region (3'-UTR) in controlling Topo II mRNA accumulation. Reporter assays in stably transfected cells demonstrated that, similar to endogenous Topo II mRNA levels, the mRNA levels of reporter genes containing the Topo II 3'-UTR varied during the cell cycle and were maximal in S and G(2)/M relative to G(1). Topo II 3'-UTR sequence analysis and RNA-protein binding assays identified a 177-nucleotide (base pairs 4772-4949) region containing an AUUUUUA motif sufficient for protein binding. Multiple proteins (84, 70, 44, and 37 kDa) bound this region, and the binding of 84- and 37-kDa (tentatively identified as the adenosine- or uridine-rich element-binding factor AUF1) proteins was enhanced in G(1), correlating with decreased Topo II mRNA levels. The binding activity was enhanced in cellular extracts or cells treated with thiol-reducing agents, and increased binding correlated with decreased Topo II mRNA levels. These results support the hypothesis that cell cycle-coupled Topo II gene expression is regulated by interaction of the 3'-UTR with redox-sensitive protein complexes.
Goswami
P C
PC
Radiation Oncology Center, MIR, Washington University Medical School, St. Louis, Missouri 63108, USA.
Sheren
J
J
Albee
L D
LD
Parsian
A
A
Sim
J E
JE
Ridnour
L A
LA
Higashikubo
R
R
Gius
D
D
Hunt
C R
CR
Spitz
D R
DR
D020413:3' Untranslated Regions / Q000235:genetics*; D016475:3T3 Cells; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology*; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D004250:DNA Topoisomerases, Type II