Nucleic Acids Research, 1988, Vol. 16, No. 6 2389-2409
© 1988
Articles |
In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site
Unité 184 de Biologie Moléculaire et de Génie Génétique de I'INSERM, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique, Faculté de Médecine 11 rue Humann, 67085 Strasbourg Cédex, France
Received February 4, 1988. Accepted February 29, 1988.
During the analysis of the in vitro alternative splicing of the natural E1A transcript of adenovirus, other minor reactions were detected (Schmitt et al., 1987, Cell 50, 31–39). We report here their characterization. The first reaction concerns the excision of a 216 nucleotide intron delineated by the 9S 5' splice site and a 3' splice site 216 nucleotides downstream. It can occur on the premRNA transcript and the 13S and 12S mRNA species. Strikingly, the reaction uses one of 3 branch points located 51, 55 or 59 residues upstream of the 3' splice site, a distance which is unusually long since all the branch points mapped up to now are located between 18–37 nucleotides of the 3' splice site. The dramatic accumulation of the corresponding lariat intermediates, likely related to this long spacing indicates that the second splicing step is relatively unefficient. The second kind of reaction analysed is a cryptic splicing which uses a 3' splice site generated by the junction of the 13S mRNA exons, and leads to the formation of 
12S and 9S mRNAs. In vitro this reaction occurs only from a 13S mRNA transcript, and not from the 13S mRNA newly formed in the splicing assay, consistent with what has been observed in vivo Thus, both the well known alternative and the minor reactions occurring in vivo from E1A premRNA and mRNAs are detected in vitro implying that most of the alternative splicing machinery is reconstituted in the in vitro system.
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