Assembly of the cleavage and polyadenylation apparatus requires about 10 seconds in vivo and is faster for strong than for weak poly(A) sites.
We have devised a cis-antisense rescue assay of cleavage and polyadenylation to determine how long it takes the simian virus 40 (SV40) early poly(A) signal to commit itself to processing in vivo. An inverted copy of the poly(A) signal placed immediately downstream of the authentic one inhibited processing by means of sense-antisense duplex formation in the RNA. The antisense inhibition was gradually relieved when the inverted signal was moved increasing distances downstream, presumably because cleavage and polyadenylation occur before the polymerase reaches the antisense sequence. Antisense inhibition was unaffected when the inverted signal was moved upstream. Based on the known rate of transcription, we estimate that the cleavage-polyadenylation process takes between 10 and 20 s for the SV40 early poly(A) site to complete in vivo. Relief from inhibition occurred earlier for shorter antisense sequences than for longer ones. This indicates that a brief period of assembly is sufficient for the poly(A) signal to shield itself from a short (50- to 70-nucleotide) antisense sequence but that more assembly time is required for the signal to become immune to the longer ones (approximately 200 nucleotides). The simplest explanation for this target size effect is that the assembly process progressively sequesters more and more of the RNA surrounding the poly(A) signal up to a maximum of about 200 nucleotides, which we infer to be the domain of the mature apparatus. We compared strong and weak poly(A) sites. The SV40 late poly(A) site, one of the strongest, assembles several times faster than the weaker SV40 early or synthetic poly(A) site.
Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095-1569, USA.
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LC
Chao
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Jamil
S J
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Kim
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Huang
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Martinson
D020413:3' Untranslated Regions / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D039221:mRNA Cleavage and Polyadenylation Factors
D020413:3' Untranslated Regions; D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D012333:RNA, Messenger; C039919:RNA, recombinant; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D012313:RNA; D011065:Poly(ADP-ribose) Polymerases