Identification ofa Specific ExonSequence ThatIsa Major Determinant intheSelection between a Natural anda Cryptic 5'Splice Site

Thefirst intron oftheearly region 3fromadenovirus type2contains a cryptic 5'splice site, Dcrl,74 nucleotides downstream fromthenatural site DI.Thecryptic site can beactivated whenthenatural site is inactivated bymutagenesis. Toinvestigate thebasis forselection between anatural andacryptic 5'splice site, we searched forcis-acting elements responsible fortheexclusive selection ofthenatural site. We showthatboth therelative intrinsic strength ofthesites andthesequencecontext affect theselection. A 120-nucleotide segment located atthe3'endofexon 1enhances splicing attheproximal site DI;initsabsence thetwosites are used according totheir strength. Thus,three cis-acting elements areinvolved inthesilencing ofthecryptic site: the sequenceofDl,thesequenceofDcrl, andan upstream exonic sequence.Weshowthat theexonic element folds, insolution, into a 113-nucleotide-long stem-loop structure. We proposethat this potential stem-loop structure whichislocated 6nucleotides upstream oftheexon 1-intron junction isresponsible forthepreferential useof thenatural 5'splice site.