Role of the branch site/3'-splice site region in adenovirus-2 E1A pre-mRNA alternative splicing: evidence for 5'- and 3'-splice site co-operation.
The adenovirus E1A gene encodes five overlapping mRNAs which are processed by alternative RNA splicing from a common pre-mRNA. To characterize cis-acting sequence elements which are of importance for the alternative 5'-splice site selection deletion and substitution mutants within the intron that is common to all E1A mRNAs were constructed. Deletion of the wild-type E1A branch site/polypyrimidine tract resulted in activation of a functionally redundant sequence located within an A/T rich sequence just upstream of the normal E1A lariat branch site. Removal of both regulatory sequences abolished in vivo splicing completely and did not lead to activation of cryptic 3'-splice sites at other locations in the E1A pre-mRNA. Furthermore we show that the sequence around the E1A branch site/3'-splice site region may have a more direct effect on the efficiency by which the alternative E1A 5'-splice sites are selected. Replacing the E1A branch site/3'-splice site region with the corresponding sequence from the second intron of the rabbit beta-globin gene or the first intron of the major late transcription unit resulted in drastic changes in E1A 5'-splice site selection. For example, with the E1A/beta-globin hybrid gene the 9S mRNA became the most abundant E1A mRNA to accumulate. This contrasts with the wild-type E1A gene in which almost undetectable levels of 9S mRNA were produced in transient expression assays. Our results strongly suggest that a cooperative interaction between 5'- and 3'-splice sites on a pre-mRNA determines the outcome of alternative splicing.
Ulfendahl
P J
PJ
Department of Medical Genetics, BMC, Uppsala, Sweden.
Kreivi
J P
JP
Akusjärvi
G
G
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*
D011743:Pyrimidines; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral