The role of the mammalian branchpoint sequence in pre-mRNA splicing.
We show that base substitutions in the mammalian branchpoint sequence (BPS) YNCUGAC dramatically reduce the efficiency of pre-mRNA splicing in vitro and alter 3' splice-site selection in vivo. Contrary to current dogma that an adenine residue at the appropriate distance from the 3' splice site is the primary determinant of lariat formation, we find that many mutations in the BPS virtually abolish splicing even though the position of this adenine is unchanged. Comparison of the analogous single-base changes in the mammalian and yeast BPSs revealed similar relative effects on splicing efficiency. However, in contrast to yeast, mammalian branchpoint mutations that decrease splicing efficiency severely do not prevent spliceosome assembly. Thus, mutations in the mammalian BPS appear to uncouple spliceosome assembly from cleavage at the 5' splice site and lariat formation.
Reed
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Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Maniatis
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D000225:Adenine / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008322:Mammals / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D000225:Adenine