Accumulation of a novel spliceosomal complex on pre-mRNAs 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 sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of 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, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent 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, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.
Champion-Arnaud
P
P
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gozani
O
O
Palandjian
L
L
Reed
R
R
D001483:Base Sequence; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000032:analysis; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D000072668:Splicing Factor U2AF
D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF