GENETIC SELECTION FOR BALANCED RETROVIRAL SPLICING - NOVEL REGULATIONINVOLVING THE 2ND STEP CAN BE MEDIATED BY TRANSITIONS IN THE POLYPYRIMIDINE TRACT

Incomplete splicing is essential for retroviral replication; and in si mple retroviruses, splicing regulation appears to occur entirely in ci s. Our previous studies, using avian sarcoma virus, indicated that wea k splicing signals allow transcripts to escape the splicing pathway. W e also isolated a series of avian sarcoma virus mutants in which env m RNA splicing was regulated by mechanisms distinct from those of the wi ld-type virus. In vitro splicing experiments with one such mutant (ins ertion suppressor 1 [IS1]) revealed that exon 1 and lariat-exon 2 inte rmediates were produced (step 1) but the exons were not efficiently li gated (step 2). In this work, we have studied the mechanism of this se cond-step block as well as its biological relevance. Our results show that the second-step block can be overcome by extending the polypyrimi dine tract, and this causes an oversplicing defect in vivo. The requir ement for regulated splicing was exploited to isolate new suppressor m utations that restored viral growth by down-regulating splicing. One s uppressor consisted of a single U-to-C transition in the polypyrimidin e tract; a second included this same change as well as an additional U -to-C transition within a uridine stretch in the polypyrimidine tract. These suppressor mutations affected primarily the second step of spli cing in vitro. These results support a specific role for the polypyrim idine tract in the second step of splicing and confirm that, in a biol ogical system, uridines and cytosines are not functionally equivalent within the polypyrimidine tract. Unlike the wild-type virus, the secon d-step mutants displayed significant levels of lariat-exon 2 in vivo, suggesting a role for splicing intermediates in regulation. Our result s indicate that splicing regulation can involve either the first or se cond step.