Small sequence insertions within the branch point region dictate alternative sites of lariat formation in a yeast intron.
The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.
Castanotto
D
D
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Rossi
J J
JJ
D017398:Alternative Splicing; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D007438:Introns; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors