A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals.
The earliest detectable mammalian prespliceosome complex (E) contains the non-snRNP splicing factor U2AF, U1 snRNP, and several spliceosome-associated proteins (SAPs). We show that specific complexes, designated E3' and E5', assemble independently on RNAs containing only a 3' or 5' splice site, respectively. U2AF is enriched in E3', whereas U1 snRNP is enriched in E5'. Using a highly sensitive substrate-competition assay, we show that both the 5' splice site and the pyrimidine tract at the 3' splice site are required for efficient E complex assembly on intact pre-mRNA. We conclude that the 5' and 3' splice sites are associated functionally as early as E complex by either direct or indirect interactions between U1 snRNP and U2AF. Our observations predict that E complex assembly is a major control point for establishing splice site selection in both constitutively and alternatively spliced pre-mRNAs.
Harvard Medical School, Boston, Massachusetts 02115.
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D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D005816:Genetic Complementation Test; D008322:Mammals / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011506:Proteins / Q000032:analysis; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics; D017381:Spliceosomes / Q000502:physiology*; D000072668:Splicing Factor U2AF
D009687:Nuclear Proteins; D011506:Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF