RNA splicing permits expression of a maize gene with a defective Suppressor-mutator transposable element insertion in an exon.

Abstract

The bz-mJ3CS9 allele of the bronze-1 gene in maize contains a 902-base-pair defective Suppressor-mutator (dSpm) transposable element in the second exon. Nevertheless, 40-50% of the enzymatic activity conditioned by a nonmutant allele at the bronze-1 locus is routinely recovered in crude extracts prepared from plants carrying bz-mJ3CS9 in the absence of an autonomous Suppressor-mutator element. Analyses of RNAs produced by such plants show that transcription proceeds through the dSpm. The dSpm sequence of the messenger RNA precursor is then removed by RNA splicing using the donor site of the single bronze-i intron and an acceptor site within the inverted terminal repeat of the dSpm. This results in a messenger RNA with the proper reading frame that could produce a functional enzyme. These data demonstrate that this dSpm insertion in an exon of a structural gene has produced a functional allele with a novel intron consisting, in part, of the dSpm. This mechanism appears to allow dSpm elements to reduce the impact of their insertions on gene expression.The maize defective Suppressor-mutator (dSpm) transposable elements are nonautonomous members of the Suppressor-mutator (Spm; also known as Enhancer-Inhibitor, En-I) family (1-3). They can transpose or excise only in the presence of an autonomous Spm element. One of the interesting features of dSpm elements, first noted by McClintock (2,4), is that association of these elements with structural genes in some cases gives rise to nonmutant phenotypes in the absence of Spmn. In order to understand the underlying mechanism of this phenomenon, we have analyzed an allele of this type at the maize bronze-1 locus.The bronze-1 locus is one of the many loci involved in the anthocyanin biosynthetic pathway of maize. The gene encodes UDP-glucose :flavonoid 03-D-glucosyltransferase (UFGTase, EC 2.4.1.91; refs. 5 and 6). Recessive alleles at the locus result in the bronze coloration of the aleurone layer and the brown coloration of many other plant parts as compared with the wild-type purple coloration. The mutant allele analyzed in the present study has a dSpm insertion in Bz, a nonmutant allele at the bronze-1 locus (7). This mutant allele has been designated bz-ml3CS9 (abbreviated as CS9). In the presence of an active Spm, expression of CS9 is suppressed in most cells except for infrequent revertant cell lineages. As a result, one observes small colored sectors on a bronze background. In the absence of an active Spmn, CS9 conditions full anthocyanin pigmentation.In this paper, we report that CS9 conditions a relatively high level of UFGTase activity in the absence of Spin despite the presence of the dSpm element in the second exon. Analyses of RNAs produced by CS9 in the absence of Spm show that the dSpm element contains an acceptor site for RNA splicing within its inverted terminal repeat. This enables RNA splicing to efficiently remove the dSpm sequence from the mRNA precursor, allowing for gene function.

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals.

“…(ii) In the case of a cauliflower mosaic virus strain which contains a mutation resulting in a partial processing of the cauliflower mosaic virus 35S RNA (in the wild-type strain the 35S RNA does not appear to be spliced), it is one of the most A+U-rich RNA regions that is spliced out (18). (iii) Transposable element sequences located in the coding regions of different maize genes are frequently spliced out, partially restoring a wild-type phenotype (21,44,47,51). In most instances, the A+U content of these sequences is high.…”

Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.

“…Earlier work with bzl-ml3 and its derivatives suggested that AS-1 splicing produced functional enzyme (Kim et al, 1987). This was based on the observation that splicing at AS-1 maintains the open reading frame.…”

“…Sequences from CS-9 (Kim et al, 1987) and bzl-R (Ralston et al, 1988) have been reported. Alleles used for flavonol 03-glucosyltransferase (UFGT) assays were in a genetic background containing B and PI to boost expression of BZ7 in husk tissue.…”

“…Splice sites in bzl-ml3, CS-3, and CS-9 were determined by RNA gel blots, analysis of cDNA clones, and S1 nuclease mapping (Kim et al, 1987;Raboy et al, 1989). These results were used to design nuclease protection experiments to quantify relative amounts of transcripts in these alleles.…”

“…These alleles condition increased or decreased enzymic activities relative to bzl-m13 (Raboy et al, 1989). Previous work determined that bzl-m13 had 13% of nonmutant enzymic activity in husk tissue (Kim et al, 1987;Raboy et al, 1989), and that splicing at AS-1, but not AS-2, produces an in-frame transcript that may encode the functional enzyme, flavonol 03-glucosyltransferase (UFGT EC 2.4.1.91). This suggests that these alleles possess splicing mutations in bzl-m13 acting to increase or To whom correspondence should be addressed at the Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611.…”