Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron.
The 3' splice site of the budding yeast Kluyveromyces lactis actin gene (ACT) intron is distally spaced (122 nucleotides) from its branchpoint and is also preceded by a silent PyAG located 43 nucleotides upstream. We devised a genetic screen that resulted in the isolation of several randomly induced cis-acting mutations that activate the silent PyAG as a 3' splice site. These mutations fall within a region surrounding this PyAG, which can hypothetically fold into a higher-order structure. Site-directed mutational analyses demonstrate that a hairpin structure in this region is required for correct 3' splice-site selection. Analysis of the point mutations suggests that local breathing of the hairpin near the first PyAG can lead to its activation. These data demonstrate that 3' splice-site selection is not a consequence of a linear, directional scanning mechanism, but support the notion of a critical positioning requirement for 3' splice-site selection. We speculate on the possible origin of this intron-encoded structural motif, which has homology to a bacterial transposon and suggests one possible origin for alternative splicing mechanisms in higher eukaryotes.
Deshler
J O
JO
Department of Microbiology, University of California, Los Angeles 90024.
Rossi
J J
JJ
D001482:Base Composition; D001483:Base Sequence; D015966:Gene Expression Regulation, Fungal; D007438:Introns; D007716:Kluyveromyces / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger