Nucleic Acids Research: VOLUME 40 ISSUE 13 2012

The adenovirus ElA region encodes three overlapping mRNAs, designated 9S, 12S and 13S. They d i f fer from each other with regard to the length of the Intron which Is removed by RNA splicing. We have constructed ElA genes with deletions and Insertions In the Intervening sequence that Is comnon to al l three ElA mRNAs, 1n a search for signals which Influence splicing of the 13S mRNA. Mutant plasmids were transfected Into HeLa cel ls and the transiently expressed ElA mRNAs characterized by the SI protectionassay. The results show that f ive upstream and 20 downstream nucieotides are suff ic ient to allow for a correct u t i l i za t ion of the 5'-spl1ce Junction for the ElA 13S mRNA. Moreover, we show that a minimal Intron length of 78 nucleotides Is required for e f f ic ient 13S mRNA splicing. The ab i l i ty of mutants with large Intron deletions to maturate a 13S mRNA could par t ia l ly be restored by expanding the Intron length with phage lambda sequences. However, 1n no case was the normal splicing eff iciency obtained with these mutants. In contrast, one mutant In which sequences from the autenthic 13S mRNA Intron were used to expand the Intron expressed almost normal levels of 13S mRNA, thus suggesting that signals which specif ical ly promote 13S raRNA splicing exist . INTRODUCTION Studies of animal DNA viruses, l i ke the human adenoviruses, have In a very Important way contributed to our current understanding of the structure and expression of eucaryotic genes. For example, a phenomenon l ike RNA splicing was f i r s t discovered by studies of adenovirus mRNA (1 ,2 ) and later shown to be a characteristic shared by eucaryotes In general ( 3 ) . The development of soluble extracts that correctly excise Introns from mRNA precursors (pre-nRNAs) has led to a dramatic Increase In our understanding of the mechanism of RNA splicing. Cofactors required for splicing have been characterized (4 ,5 ,6) and some remarkable Intermediates which are formed during the splicing process have also been Identi f ied ( 7 , 8 , 9 ) . However, the signals that direct the excision of Intron sequences from pre-mRNAs are s t i l l poorly characterized. Consensus sequences have been derived for both 5 ' and 3' -spl ice junctions (3,10) and snail changes In and around the conserved GT © IR L Press Limited, Oxford, England. 6 2 9 9 Nucleic Acids Research