ACCUMULATION OF A NOVEL SPLICEOSOMAL COMPLEX ON PRE-MESSENGER-RNAS CONTAINING BRANCH SITE MUTATIONS

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint seq uence (BPS) have no apparent effect on E complex formation but block t he assembly of the A complex and the UV cross-linking of U2 small nucl ear ribonucleoprotein particle (snRNP) proteins, Unexpectedly, a novel complex, designated E, assembles on pre-mRNAs containing BPS mutatio ns. Unlike the E complex, the E complex accumulates in the presence o f ATP, U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E complex, Significantly, prev ious work showed that U1 snRNP and U2AF become destabilized from pre-m RNA after E complex assembly on normal pre-mRNAs, Thus, our data are c onsistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E-->A). In the first step, U 1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction, In the second step, the stable binding of U2 snRNP occurs i n a BPS-dependent reaction.