Splicing of adenovirus 2 early region 1A mRNAs is non-sequential.

The r-strand of early region 1A (E1A) of adenovirus serotype 2 is transcribed into three completely overlapping messenger RNA species, a 9S, a 12S and a 13S mRNA. These three mRNAs are processed from a common colinear RNA precursor and differ only with regard to the size of the intron removed during mRNA maturation. We have studied the processing pathways for the E1A mRNAs by using an assay for transient expression of recombinant plasmids containing the E1A region. All three region E1A mRNAs are synthesized and transported to the cytoplasm in sufficient quantities to permit a detailed study of their structure by S1 endonuclease analysis and primer extension. Additionally, we show that the 72 base-pair repeat from simian virus 40 (SV40), when located upstream of the E1A promoter stimulates expression of the E1A mRNAs five- to tenfold. In order to determine whether splicing of the E1A mRNAs is sequential, i.e. whether the 13S and 12S mRNAs can serve as intermediates in splicing, we constructed two plasmids that lack the intervening sequences that are removed during the maturation of the 12S and 13S mRNAs, respectively. From an analysis of the RNAs produced after transfection with these deletion mutants, the following major conclusions can be made. (1) Splicing of the E1A mRNAs is non-sequential, i.e. the 13S, 12S and 9S RNAs are generated by separate splicing events using the nuclear colinear transcript as the only precursor RNA. (2) RNA splicing is not a prerequisite for an efficient transport of the E1A mRNAs to the cytoplasm. (3) The 13S RNA can be further processed to 12S and 9S RNA species. These splicing events are, however, illegitimate and give rise to 12S and 9S RNAs that both lack one nucleotide at the splice junction. (4) A coupling between splicing and nuclear transport is most likely required in vivo to prevent illegitimate splicing of the 13S mRNA. (5) The 12S RNA does not serve as a precursor for further processing to the 9S RNA. (6) Splicing of the E1A mRNAs followed strictly the G-T-A-G rule.