Nuleclear extracts of Crithidia fasciculata contain a factor(s) that binds to the 5'-untranslated regions of TOP2 and RPA1 mRNAs containing sequences required for their cell cycle regulation.
The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene, TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5'-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5'-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP2 5'-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1 mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5'-UTR of their transcripts.
Mahmood
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R
Molecular Biology Institute and Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90095-1570, USA.
Ray
D S
DS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D016384:Consensus Sequence; D016831:Crithidia fasciculata / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051716:Replication Protein A; D016196:S Phase; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D051716:Replication Protein A; D004250:DNA Topoisomerases, Type II