Characterization of human RNA splice signals by iterative functional selection of splice sites

An iterative in vitro splicing strategy was employed to select for optimal 3' splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the s elected sequences contained a conserved AAUAAAG motif that proved to be fun ctional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the <(AA)under bar>U<(AA)under bar>AG sequence revealed that the branching process proceeded efficiently but quit e promiscuously. Surprisingly, the conserved guanosine, adjacent to the com mon AAUAAA polyadenylation motif, was found to be required only for polyade nylation. In an independent experiment, sequences surrounding an optimal br anchpoint sequence were selected from two randomized 20-nt regions. The clo nes selected after six rounds of splicing revealed an extended polypyrimidi ne tract with a high frequency of UCCU motifs and a highly conserved YAG se quence in the extreme 3' end of the randomized insert. Mutating the 3' term inal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.