A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation

Abstract

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.

Figure 1

RT-PCR analysis of mutant bt2-7503 transcript. A, Total endosperm RNA from the wild type and from bt2-7503 was subjected to RT-PCR using primers spanning exons 1 to 10 of the Bt2 transcript. Products were resolved on a 1% agarose gel, blotted, and probed with the full-length Bt2 cDNA probe. Fragments from the mutant were excised and sequenced. Schematic representations of the bt2-7503 RT-PCR products are shown. *, Region missing nine nucleotides from the 3′ portion of exon 3. B, Schematic representation of the genomic sequence bearing the splice site alteration of the mutant bt2-7503. Exon and intron sequences are given in capital and small letters, respectively. The mutant splice site nucleotide in intron 3 is marked by a downward arrow. Arrows joined by lines mark the donor and acceptor site utilized during splicing. #, Intron 2 is spliced normally in type II transcript; *, intron 3 is not spliced in type I transcript.

Figure 2

RNA blots of wild-type and bt2-7503 RNA. Total 20-d post-pollination endosperm RNA was subjected to electrophoresis, blotting, and hybridization with exons 1 to 3 of Bt2 cDNA (left). The blot was stripped and re-probed with exons 4 to 10 (middle) and then with the full-length Bt2 cDNA probe (right). Molecular mass markers are shown on the left.

Figure 3

Branch point mapping strategy and the structure of the intron lariat intermediate. A, The RT-PCR strategy employed for mapping branch point sequence is outlined. The intron 3 lariat is represented by a blue line. Red arrows represent the positions of the primers used during RT-PCR. First-strand DNA synthesized with primer Lo1 is represented by a broken blue line. Primers Lo2 and Up1 were employed for subsequent amplification. B, Schematic representation of the lariat intermediate during processing of mutant bt2-7503 transcript. The regions corresponding to exons 1 to 3 and to intron 3 in the form of a lariat attached to the downstream exons are shown. Informative sequences of the intron lariat and of exon 4 are presented. Exon and intron sequence are in capital and small letters, respectively. The G residue at the −23 position of exon 3, which forms a covalent linkage with branch point residue A at −23 position of intron 3, is shown in bold type. Residue a at the 5′ end of intron 3 is underlined.

Figure 4

Mapping of the branch point nucleotide during the processing of the mutant bt2 transcript. A, The genomic sequence spanning exon 3 to exon 4 of the mutant bt2-7503 is given with exons in uppercase letters and intron sequences in lowercase letters. Primers used during RT-PCR are underlined with arrows. The mutated 5′ end nucleotide of intron 3 is underlined and the branch point nucleotide is boxed. B, Ethidium-bromide-stained gel of the RT-PCR product resolved on 1% agarose gel resulting from subjecting total bt2-7503 endosperm RNA to RT-PCR as outlined in A. The size of the product is shown on the left. Marker mass markers are given on the right.