In vitro formation of a lariat structure containing a G2'-5'G linkage.
We have examined the effects of base changes at the lariat branch site of a modified adenovirus major late precursor mRNA (pre-mRNA). Replacement of the A residue at the lariat attachment site with a G residue was studied. Incubation of this altered pre-mRNA with nuclear extracts of HeLa cells yielded less spliced mRNA (10-fold) than similar reactions with the wild type pre-mRNA. The intron lariat formed during the reaction with the mutant transcript contained the predominant branch (2'-5' phosphodiester linkage) to an upstream A residue. In contrast, the intron/exon 2 lariat contained the predominant branch to the substituted G residue. These results indicated that detectable spliced RNA was formed when the lariat was attached at the A residue but not when the lariat was attached to the substituted G residue. A second mutation was introduced into the transcript by substituting an additional G residue at the alternative A branch site. When transcript derived from this plasmid was incubated with nuclear extract, cleavage occurred at the 5' splice site, and an intron/exon 2 lariat was produced, but spliced RNA was not detected. T1 RNase digestion and primer extension analyses of this intron/exon 2 lariat revealed that all of the lariat formed on the G residue at the normal attachment site.
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D005091:Exons; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007438:Introns; D009690:Nucleic Acid Conformation; D009694:Nucleic Acid Precursors / Q000096:biosynthesis*; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012322:RNA Precursors; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D006163:Ribonuclease T1 / Q000378:metabolism
D009694:Nucleic Acid Precursors; D009841:Oligonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D012313:RNA; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme; D006163:Ribonuclease T1