IN-VIVO SELECTION FOR INTRONIC SPLICING SIGNALS FROM A RANDOMIZED POOL

Retroviruses utilize balanced splicing to express multiple proteins fr om a single primary transcript. A number of cis-acting signals help ma intain this balance, including the branch point sequence (BPS), polypy rimidine tract (PPyT) and sequences within the downstream exon. In gen eral, regulated splicing requires weak splicing signals and we have pr eviously shown the same requirement for the simple retrovirus, avian s arcoma virus (ASV), Here we take advantage of the requirement for bala nced splicing in retroviral replication to examine the sequence constr aints of an intronic splicing element. Selection for replication compe tence makes it possible to amplify and identify functional sequences f rom a pool of all possible sequences. In this report we examine the ro le of pyrimidines within the PPyT, Our results provide in vivo confirm ation that the functional strength of a PPyT is related to its length and uridine content and that the PPyT plays a role in the second step of the splicing reaction. We also show that the minimal distance betwe en the 3'-splice site and the BPS in this system is 16 nt. With modifi cation, the selection system described here can be used to examine the sequence constraints of other exonic or intronic splicing elements in vivo.