ROLE OF THE CONSTITUTIVE SPLICING FACTORS U2AF(65) AND SAP49 IN SUBOPTIMAL RNA SPLICING OF NOVEL RETROVIRAL MUTANTS

Retroviruses display a unique form of alternative splicing in which bo th spliced and unspliced RNAs accumulate in the cytoplasm, Simple retr oviruses, such as avian sarcoma virus, do not encode regulatory protei ns that affect splicing; this process is controlled solely through int eractions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel a vian sarcoma virus mutants. These viruses were separated into two clas ses based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants wi th altered polypyrimidine tract or branch point sequences, showed sign ificant accumulation of intermediates, suggesting that splicing was re gulated in step 2, The other class, which included mutants with deleti ons of exonic enhancer sequences, did not accumulate splicing intermed iates, suggesting that splicing was regulated before step 1 of the spl icing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last splic eosomal complex. Using UV cross-linking methods, we show that regulati on at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with dec reased crosslinking of the factor U2AF(65), whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49, Beca use these mutations were isolated by selection for replication-compete nt viruses, we conclude that retroviral splicing may be regulated in v ivo through altered binding of constitutive splicing factors.