SEQUENCE REQUIREMENTS FOR RNA SPLICING IN THE E1A GENE OF ADENOVIRUS TYPE 5 (EUKARYOTE)
Abstract
Most eukaryotic genes examined to date contain internal sequences that are present in the DNA but not in their derived mature RNA. A process known as RNA splicing has been implicated in the removal of the internal sequences during RNA maturation. Although a great deal of attention has been paid to the process of RNA splicing, little is actually known regarding its regulation and mechanism. Conserved sequences have been found in pre-mRNA molecules. These include the consensus donor site at the 5' junction between exon and intron, the consensus acceptor site at the 3' junction between intron and exon, and a consensus branch point located within the intron. This last region is located 15 to 40 nucleotides upstream from the 3' acceptor site and has rather limited sequence requirements. Besides these three regions, no required sequences have been identified. Yet these sequences are probably not all that are required for splicing to occur. Donor and acceptor sites appear in the genome of eukaryotes at a higher frequency than they are utilized, so other factors must be involved in regulating the process. Presumably these factors require binding to regions of the RNA precursors. To investigate this possibility, systematic deletions of the exons and intron of the adenovirus E1A gene have been carried out to determine the minimum sequences necessary to correctly splice the precursor RNA. It was found that a only 4bp of 5' exon immediately upstream from the donor site and 3bp of 3' exon immediately downstream from the acceptor site were required for correct splicing. The E1A gene is capable of alternative splicing, and intron deletions had different effects on the two possible pathways. The 13s intron showed a size dependency for splicing, while the larger 12s intron was unable to splice correctly in two deletion mutants, even though the size of the intron was still larger than the 12s wild type intron. This indicated that size, while important, was not the only limiting factor in E1A pre-mRNA splicing.
Degree
Ph.D.
Subject Area
Molecular biology
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