A Splice Site Mutant of Maize Activates Cryptic Splice Sites, Elicits Intron Inclusion and Exon Exclusion, and Permits Branch Point Elucidation

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5′ terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5′ to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5′ to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030–2031) led to the identification of 3′ intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing.