Sequence-specific endonucleolytic cleavage and protection of mRNA in Xenopus and Drosophila.
Previously, a small region of the 3'-untranslated region (3' UTR) of Xlhbox2B mRNA was shown to be sufficient for sequence-specific endonucleolytic cleavage after injection into Xenopus oocytes. Here, we report an in vitro RNA degradation reaction that mimics the in vivo result accurately. The reaction also reveals that oocytes contain a sequence-specific RNA-binding factor that inhibits the endoribonuclease. These opposing activities may be regulated during Xenopus oogenesis. Partial purification shows that the endonuclease does not require translation or ribosomes and does not resemble previously described RNA processing complexes. We have isolated another Xenopus cDNA, Xoo1, that contains a long, repetitive destabilizing element similar to the one in Xlhbox2B. Based on a comparison of these natural destabilizing sequences and in vitro mutagenesis experiments, we find that a single destabilizing site is, at most, 19 bases in length and that the endonuclease and protective factor recognition sites may be overlapping subsets of this sequence. Finally, we show that Drosophila embryos contain similar activities, each of which can use Xenopus recognition sites. This level of conservation suggests an important biological function for this system of post-transcriptional regulation.
Brown
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BD
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Zipkin
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Harland
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017124:Conserved Sequence; D004331:Drosophila melanogaster / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000201:enzymology; D004722:Endoribonucleases / Q000378:metabolism*; D006207:Half-Life; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000201:enzymology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000235:genetics
D012333:RNA, Messenger; D004722:Endoribonucleases