INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Nonsense-mediated decay (NMD) is a mechanism in which abnormal mRNAs containing premature translation termination codons are efficiently eliminated so that production of undesirable truncated proteins is avoided (for review, see Maquat, 1995; Culbertson, 1999; Hentze and Kulozik, 1999). This RNA surveillance system is universally present in eukaryotes, and, in particular, it has been extensively studied in yeast and mammals (for review, see Maquat, 1995; Culbertson, 1999; Hentze and Kulozik, 1999). In mammals, several features of NMD have been recently revealed. First, although the majority of mammalian mRNAs are subject to NMD prior to release from their association with nuclei (nucleus-associated NMD), some mRNA are exclusively subject to NMD in the cytoplasm. (Moriarty et al., 1998; Culbertson, 1999; Hentze and Kulozik, 1999; Sun et al., 2000). Second, splicing of at least one intron is required for NMD to occur (Moriarty et al., 1998; Zhang et al., 1998a, 1998b). This observation suggests a link between splicing in the nucleus and actual RNA degradation in the cytoplasm. The current model for NMD in mammalian cells proposes that splicing leaves behind a mark at 3'-most exon-exon junction by proteins stably associated with mRNA after spliceosome dissociation and that positions of premature translation termination codons relative to this mark are important concerning whether or not mRNA is subject to NMD (Thermann et al., 1998; Hentze and Kulozik, 1999; Le Hir et al., 2000; Sun et al., 2000). Third, NMD is dependent on the termination codon position; if translation terminates >50 to 55 nucleotides upstream of the 3'-most exon-exon junction, NMD occurs (Thermann et al., 1998; Zhang et al., 1998a, 1998b; Sun et al., 2000).

In yeast, the presence of a downstream instability element has been found, marking the position of the translation termination codons required for NMD. If translation termination codons are found 3' from the downstream instability element, the mRNA is subject to NMD (Culbertson, 1999; Hentze and Kulozik, 1999). Similar elements do not appear to be present in mammalian cells, suggesting the existence of major differences in the mechanism of NMD between yeast and mammalian cells. The presence of such elements in plants remains to be studied.

Little is known about NMD in plants. Mutant alleles of a soybean Kunitz trypsin inhibitor gene (Kti3) and the phytohemagglutinin gene (PHA) from common bean (Phaseolus vulgaris), which produce mRNAs containing premature termination codons, have been shown to be more quickly degraded than those of the wild-type genes (Jofuku et al., 1989; Voelker et al., 1990). In the case of the pea ferredoxin gene (FED1), its expression is posttranscriptionally regulated by light (Dickey et al., 1994), and insertion of stop codons in the coding region causes a decrease in mRNA stability in the light but not in the dark (Dickey et al., 1994; Petracek et al., 2000). Furthermore, the effect of premature translation termination codons on the stability of FED1 mRNA depends on their positions, and this mRNA degradation pathway is different from that operating in the dark (Petracek et al., 1998). The position-dependent effect of translation termination codons on mRNA stability has been also demonstrated in bean PHA mRNA (van Hoof and Green, 1996). These studies suggest that NMD occurs in plant mRNA. However, these three genes, Kti3, PHA, and FED1, which were previously studied for an NMD-like phenomenon, are all intronless genes. Considering the observation that intron splicing is an important step in the NMD pathway in mammalian cells, studies need to be performed with intron-containing plant genes to better understand this phenomenon in plants.

It was very recently shown in Caenorhabditis elegans that NMD is linked with RNA interference (RNAi) caused by dsRNA (Fire et al., 1998) by the analysis of the effects of smg mutations originally isolated as mutations affecting NMD on the initiation and maintenance of RNAi (Domeier et al., 2000). Because RNAi and posttranscriptional gene silencing (PTGS), which are widely observed in plants, are thought to share some molecular mechanisms (Hamilton and Baulcombe, 1999; Hammond et al., 2000; Morel and Vaucheret, 2000; Zamore et al., 2000), it is becoming increasingly important to study the NMD phenomenon in plants to better understand the epigenetic silencing caused at the level of RNA.

In this study, we show that two mutant alleles of the rice (Oryza sativa) waxy gene cause decreased accumulation of the fully spliced RNA but not of the RNA retaining intron 1. The sequencing of these alleles showed that, in each of the two mutant genes, a premature termination codon is created by mutations. Measurements of the half-lives of unspliced and spliced RNAs containing the identical premature termination codon indicated that the fully spliced RNA is degraded much faster than the partially spliced RNA. These results indicate that NMD does occur in plant mRNA and that splicing may be linked with NMD, as has been observed in mammalian RNA.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Decreased Accumulation of Spliced mRNA But Not of Partially Spliced mRNA Retaining Intron 1 in Two Mutant waxy Genes of Rice

To elucidate the molecular basis of the two waxy mutations of rice in which no amylose synthesis occurs in the endosperm, we initially analyzed the waxy transcripts of the mutant endosperms by RNA gel-blot analysis. Two wild-type alleles are known at the waxy locus of cultivated rice, and the Wx^b allele is present in all Japonica cultivars (Sano, 1984). The two mutant waxy lines used for this study are both in the Japonica background. The Wx^b allele has a single-base mutation at the 5'-splice site of intron 1, and it causes accumulation of transcripts of 3.4 kb in size in which intron 1 is unspliced but the other 12 introns are fully spliced (Bligh et al., 1998; Cai et al., 1998; Hirano et al., 1998; Isshiki et al., 1998). As in our previous reports (Isshiki et al., 1998, 2000) the partially spliced 3.4-kb transcript retaining the 1.1-kb intron 1 is called unspliced RNA in this study. As shown in Figure 1, results of RNA gel-blot analysis showed that levels of spliced transcripts for both mutants, EM21 and cv Musashimochi, were approximately 20% of those in the wild type, whereas the levels of transcripts still retaining the 1.1-kb intron 1 were comparable between the wild type and the mutants. The decreased accumulation of spliced transcripts, but not of unspliced transcripts, suggested that mutant waxy transcripts are spliced either less efficiently than those of the wild type or that spliced transcripts are less stable than those of the wild type.

View larger version (51K): [in this window] [in a new window]   Figure 1.   RNA gel-blot analysis of Wxb RNAs in mutant waxy endosperms. Two different transcripts, the unspliced 3.4-kb transcript retaining the 1.1-kb intron 1 and the spliced 2.3-kb transcript, were detected in the wild type carrying the Wxb allele and two waxy mutants originated from the Wxb allele. As in our previous reports (Isshiki et al., 1998, 2000), the partially spliced 3.4-kb transcript retaining the 1.1-kb intron 1 is called unspliced RNA in this study.

Sequence Analysis of the Mutant waxy Genes Revealed the Presence of Premature Translation Termination Codons in the Coding Region

To understand the basis of the decreased RNA accumulation of the spliced mRNA in the waxy mutants, sequences of the waxy genes from 630 bp relative to the transcription start site through the 3'-untranslated region were determined for the two mutant waxy genes. Results of the sequence analysis indicated that, in the EM21 mutant, a G to A mutation in a TGG codon created a TGA termination codon in exon 7 (Fig. 2, A and B). In cv Musashimochi, duplication of the 23-bp sequence was found in exon 2, which generated a TGA stop codon within this exon (Fig. 2, A and C). No other base changes were found between the two mutants and the corresponding wild type (data not shown). Reduced accumulation of spliced transcripts in the mutant endosperms might be caused by the NMD of waxy mRNAs, since premature stop codons were found in the second and seventh exon for cv Musashimochi and EM21 waxy mutations, respectively. Studies of mammalian NMD clearly established that all premature translation termination codons that are present more than 55 nucleotides upstream of the 3'-most exon-exon cause NMD (Thermann et al., 1998; Zhang et al., 1998a, 1998b; Sun et al., 2000).

View larger version (38K): [in this window] [in a new window]   Figure 2.   Sequence changes in the waxy genes of the two mutants. A, The structure of the Wxb gene and locations of sequence alterations in the two mutants. Exons are shown by boxes and numbered 1 through 14. , The initiation and the stop codons; , the locations of mutations in cv Musashimochi and EM21. AG/tt indicates the sequences of the 5'-splice site of intron 1. B, The sequence of the 3' end of intron 6 and the 5' end of exon 7 in the EM21 mutant. C, The sequence from the region of the mutation in exon 2 of cv Musashimochi. The white box indicates duplication of the 23-bp sequence. Introns are indicated in lowercase letters, and exons are shown in capital letters. A premature nonsense codon generated by the mutation is double underlined.

The observation that there were no differences in levels of unspliced mRNA between the wild type and the mutants suggests that the NMD of waxy mRNA might be linked with splicing although the actual intracellular site of RNA degradation remains to be studied. This finding is consistent with previous observations in mammalian cells, in which splicing is required for appropriate NMD irrespective of the site of RNA degradation (Moriarty et al., 1998; Zhang et al., 1998a, 1998b; Culbertson, 1999; Hentze and Kulozik, 1999).

Alternatively Spliced mRNAs Are Subject to NMD with Similar Efficiencies

The waxy mutants used in this study were in the Japonica background, and our sequence analysis showed that they have a splice site mutation in intron 1 as does their progenitor Wx^b allele (Fig. 2A). Because there are several lines of evidence indicating that the efficiency of splicing and alternative splicing is related to NMD (Dietz et al., 1993; Lozano et al., 1994) we tested whether certain aspects of intron splicing influenced the efficiency of NMD in waxy mRNA. The rice waxy genes provide a unique in vivo situation in which NMD occurs with mRNA that has undergone alternative splicing and the efficiency of those splicing reactions is low.

First, we examined whether alternatively spliced mRNAs were subject to NMD with similar efficiencies. Alternative splicing of intron 1 in Wx^b RNAs produce three different mRNAs in the endosperms (Fig. 3A). Reverse transcriptase (RT)-PCR analysis and sequencing revealed two different mRNAs that are spliced at site 1 and one class of mRNA spliced at site 2 (Fig. 3A; Isshiki et al., 1998, 2000). Because the two mRNAs spliced at site 1 have only one nucleotide difference, they are not distinguished by RT-PCR analysis. Therefore, we examined whether mRNA spliced at site 1 or site 2 was differentially degraded by using a competitive RT-PCR analysis. Results shown in Figure 3, B and D, clearly indicated that alternatively spliced mRNAs were subject to NMD with similar efficiencies. By using the same RT-PCR method, we quantified the levels of unspliced RNAs in the wild type and the two mutants. Results indicated that the levels of unspliced waxy RNAs were similar between the wild type and the two mutants (Fig. 3, C and D) and that they were consistent with the results of the RNA-blot analysis (Fig. 1). These results suggest that alternatively spliced mRNAs are subject to NMD with similar efficiencies.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Alternatively spliced Wx mRNAs are subject to NMD with similar efficiencies in the endosperm. A, The splicing pattern of rice Wxb pre-mRNAs. Wxb has a G-to-T mutation at the 5'-splice site of intron 1 and generates two kinds of mRNAs spliced at two locations at site 1 (Isshiki et al., 1998; 2000). They are not distinguishable by RT-PCR analysis. In addition, mRNA spliced at a cryptic site (site 2), 100 nucleotides upstream of site 1, is produced. White bars indicate exons, and black bars indicate the distal part of exon 1 flanked by the site-2 splice site and the authentic 5'-splice site (site 1). When site 2 is used for splicing, transcripts become approximately 100 nucleotides shorter than those spliced at site 1.Thin bars indicate the first intron. B, Competitive RT-PCR analysis of spliced waxy transcripts in the wild type (WT) and the two mutants. C, Competitive RT-PCR analysis of unspliced waxy transcripts in the wild type (WT) and the two mutants. D, Quantitative representations of the spliced and unspliced waxy transcripts shown in B and C.

Efficiency of Intron Splicing Does Not Influence the NMD of the waxy mRNA

It is known that waxy mutations are rarely found in Indica varieties that carry the nonmutant Wx^a allele (Y. Sato, unpublished data), in which no transcripts retaining intron 1 are produced. These observations made us wonder whether or not there is a link between the inefficient splicing of intron 1 usually observed for Wx^b RNAs and NMD. To address this question, we constructed four waxy genes (Fig. 4A). Furthermore, to perform transient expression in rice protoplasts, we used the 35S promoter for these constructs. 35S Wx^b is a wild-type Wx^b having the mutant AG/TT 5'-splice site of intron 1 and no premature nonsense codon. In contrast, 35S Wx^b Stop has a premature nonsense codon in exon 7 as does the EM21 mutant. The nonmutant AG/GT was introduced to these two constructs to make the 35S Wx^a and 35S Wx^a Stop. These constructs were transfected into rice protoplasts by electroporation, and RNAs isolated from the protoplasts were subjected to a competitive RT-PCR analysis.

View larger version (43K): [in this window] [in a new window]   Figure 4.   Relationship between efficiencies of intron 1 splicing and NMD of waxy mRNA. A, Constructs used for assays. White boxes represent exons, and solid lines represent introns. 35S Wxb has a TT mutation at the 5'-splice site of intron 1, and 35S Wxb Stop has a premature stop codon in exon 7. The TT at the 5'-splice site of intron 1 present in 35S Wxb and 35S Wxb Stop was mutated to GT in 35S Wxa and 35S Wxa Stop. B, Competitive RT-PCR analysis of RNAs from 35S Wxb and 35S Wxb Stop in transfected rice protoplasts. In rice protoplasts, site 2 was preferentially used for splicing, and RNAs spliced at site 1 were not detectable. C, Competitive RT-PCR analysis of RNA from 35S Wxa and 35S Wxa Stop in transfected protoplasts. Unspliced waxy RNA was not detected because Wxa RNA was completely spliced at site 1 (Isshiki et al., 1998; 2000).

First, we compared 35S Wx^b Stop with 35S Wx^b. In rice protoplasts, site 2 was preferentially used for splicing, and transcripts spliced at site 1 were not detectable. Results indicate that spliced RNA was considerably reduced in 35S Wx^b Stop, whereas the level of unspliced RNA was comparable between 35S Wx^b and 35S Wx^b Stop (Fig. 4B). These results were essentially the same as those obtained in the endosperm, suggesting that a similar mechanism of NMD operates in the endosperm and cultured cells of rice. Next, we tested whether NMD occurs on mRNA produced from 35S Wx^a Stop by comparing mRNA produced from 35S Wx^a Stop with that from 35S Wx^a (Fig. 4C). It was noted that levels of RNAs were more than two orders of magnitude higher than those derived from 35S Wx^b as demonstrated previously (Isshiki et al., 1998). The level of mRNA obtained from 35S Wx^a Stop was approximately 25% of that of 35S Wx^a. These results clearly indicate that NMD also occurs with Wx^a mRNA, which has no mutation at the intron 1 splice site.

Decreased Half-Life of waxy mRNA Containing a Premature Nonsense Codon

To test if a premature nonsense codon decreases the stability of mRNA, the half-lives of spliced and unspliced waxy RNAs were measured by incubating protoplasts transformed with 35S Wx^b and 35S Wx^b Stop with actinomycin D, an inhibitor of transcription, for various times and quantifying their amounts by RT-PCR (Fig. 5). The half-life of spliced waxy mRNA with a premature stop codon was 5.3 min, whereas that of the spliced mRNA without a premature stop codon was 17.5 min. These results clearly indicated that the presence of premature translation termination codons decreases the stability of waxy mRNA approximately 3-fold. This value is in good agreement with the decrease in the half-life of mRNA derived from a mutant frame-shift allele of the bean PHA gene having a premature nonsense codon (van Hoof and Green, 1996). Another important finding was that unspliced RNA derived either from Wx^b or Wx^b Stop was equally stable and that no sign of degradation was detected even 20 min after actinomycin D treatment (Fig. 5). These results suggest that the NMD of the rice waxy gene operates only with those RNAs that contain premature nonsense codons and are fully spliced.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Faster decay of the spliced waxy mRNA containing a premature stop codon in transfected rice protoplasts. 35S Wxb and 35S Wxa Stop were transfected into rice protoplasts by electroporation, and levels of spliced and unspliced waxy RNAs were quantified by competitive RT-PCR analysis at various times after actinomycin D addition.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Rice waxy Mutations Provide an Opportunity to Study NMD of Plant mRNA

Rice waxy mutations provided an opportunity to study NMDof both spliced and partially spliced RNAs, since the progenitor wild-type allele of the mutants is the Wx^b allele, which generates both un-spliced and spliced RNAs due to a single-base mutation at the 5'-splice site of intron 1 (Bligh et al., 1998; Cai et al., 1998; Hirano et al., 1998; Isshiki et al., 1998). Because of this unique situation, we could clearly demonstrate that fully spliced mRNA containing a premature nonsense codon was subject to NMD, whereas the partially spliced RNAs were not degraded although they contained a premature nonsense codon at an identical position.

One interesting observation is that unspliced mRNA is stable although it carries a number of nonsense codons within intron 1 (Isshiki et al., 1998). This implies that nonsense codons present in intron 1 were not recognized by the NMD system and only those nonsense codons present downstream within the exons are recognized by the NMD system in rice. These findings raise a possibility that not only splicing but other processing steps may be involved in NMD of rice waxy mRNA.

Although we found a linkage between splicing and NMD in rice waxy mRNA, one major difference between our findings and mammalian NMD is clear. Normally splicing of an intron 3' to the nonsense codon is important for mammalian NMD, whereas in the case of rice waxy NMD, splicing of the first intron present upstream of the premature nonsense codon was important. This difference may reflect differences in the mechanisms of NMD between mammals and plants. Future studies will clarify this important issue.

In mammals, the majority of NMD occurs while mRNA is still associated with the nucleus, whereas some mRNAs are subject to NMD exclusively in the cytoplasm (Maquat, 1995; Moriarty et al., 1998; Hentze and Kulozik, 1999; Sun et al., 2000). In the case of rice waxy mRNA, NMD correlates with splicing of intron 1 although the site of NMD remains to be studied. NMD may only occur with properly processed RNAs and partially spliced RNA might be physically separated from other RNAs in the nucleus. Alternatively, if NMD of waxy mRNA exclusively occurs in the cytoplasm, lack of splicing may prevent the export of unspliced RNA to the cytoplasm for RNA decay. In this latter case splicing per se would not be an important factor for NMD.

All three plant genes whose transcripts were previously shown to be degraded due to the presence of premature nonsense codons are intronless (Jofuku et al., 1989; Voelker et al., 1990; Dickey et al., 1994; van Hoof and Green, 1996; Petracek et al., 2000). If positions of premature stop codons are recognized by proteins involved in splicing reactions and stably associated with mRNA at exon-exon junctions even after spliceosome dissociation, as postulated for mammalian NMD (Thermann et al., 1998; Hentze and Kulozik, 1999; Le Hir et al., 2000; Sun et al., 2000), a question arises asto whether degradation of RNAs containing premature stop codons derived from intronless genes occurs through the same mechanism observed in mammalian NMD. This remains to be studied.

NMD and Other Types of Posttranscriptional Regulation of Rice Wx Gene Expression

We have previously shown the other types of post-transcriptional regulation of rice waxy gene expression (Itoh et al., 1997; Terada et al., 2000). In transgenic Japonica rice carrying the antisense Wx gene, reduction of sense RNA by the antisense RNA was only observed with the spliced RNA, and no changes were found with unspliced RNA retaining intron 1 (Terada et al., 2000). This observation is very similar to observations regarding the waxy mutants described in this paper. This similarity may suggest that RNA degradation caused by antisense RNA is mechanistically related to NMD. The molecular mechanism of gene suppression by antisense genes has not been well understood (Bourque, 1995), and it is now considered that some effects are caused by RNA degradation mediated by dsRNA (for review, see Kooter et al., 1999; Meins, 2000; Morel and Vaucheret, 2000).

Introduction of the rice wild-type waxy gene into a Japonica rice causes silencing of both the endogenous waxy and the transgenes in pollen with high frequency (Itoh et al., 1997). Although this study did not examine experimentally whether the observed gene silencing in transgenic rice was post-transcriptional, the observed cosuppression of the endogenous waxy and the transgenes is consistent with a posttranscriptional mechanism. Future experiments examining both NMD and PTGS of Wx gene expression in transgenic waxy mutants may give insights into common elements in the molecular mechanisms of NMD, PTGS, and RNAi in plants.

NMD Is One of Many RNA Degradation Pathways in Plants

Regulation of mRNA stability plays a key role in the control of gene expression in plants and other eukaryotes. Various mRNA decay pathways are thought to exist in plants (Gutierrez et al., 1999). For instance, a DST (downstream element) present in the 3'UTR was shown to be an mRNA instability element of the Arabidopsis SAUR gene (Gil and Green, 1996), and adenylate/uridylate-rich elements present in some mammalian genes confer instability to reporter transcripts in transformed tobacco cells (Ohme-Takagi et al., 1993). Premature nonsense codons may be categorized into such instability elements. Therefore, the elucidation of various mRNA decay pathways and mechanistic relationships among multiple pathways will become critical in future studies. The molecular mechanisms of RNA degradation in PTGS may also be related to the above-mentioned RNA decay pathways. Rice waxy genes will be a useful experimental tool for studies in the regulation of RNA stability in plants.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material

A Japonica rice (Oryza sativa) cultivar, cv Kinmaze, a waxy mutant EM21 with the Kinmaze background (Satoh, 1986), and a glutinous rice cultivar, cv Musashimochi, were all grown in a greenhouse under a light-dark cycle consisting of 14 h of light and 10 h of dark.

RNA Gel-Blot Analysis

Total RNA was prepared from immature seeds 15 d after pollination by the published method (Chomczynski and Sacchi, 1987). RNA (10 µg) was separated by electrophoresis in 1% (w/v) agarose gels containing formaldehyde, blotted, and hybridized with a waxy cDNA probe that had been labeled with [-³²P]dCTP using the Multiprime DNA labeling system (Amersham, Buckinghamshire, UK). Probes used were a 1.3-kb BglII fragment of waxy cDNA (exon 5-exon 14). After the membrane was washed, an autoradiography was obtained by exposing the membrane to an x-ray film.

DNA Sequencing

The exon, intron, and promoter (630 bp relative to the transcription initiation site) sequences of cv Kinmaze, EM21, and cv Musashimochi were amplified by PCR from genomic DNA using pairs of Wx-specific primers. PCR products were cloned into the pGEM-T vector (Promega, Madison, WI). Sequencing of cloned products was performed with a Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PE-Applied Biosystems, Foster City, CA).

Construction of Plasmids

35S Wx^b carries the coding region of Wx^b isolated from a genomic library of cv Kinmaze under the control of the cauliflower mosaic virus 35S promoter. 5'UTR, open reading frame, and 3'UTR regions of Wx^b were separately cloned after PCR amplification and subsequently ligated together on a pSN221 vector. 35S Wx^b had the following structure with restriction sites used for ligation: 35S promoter-SalI-5'UTR-NheI-waxy ORF-HpaI-3'UTR-NotI-NOS terminator. To produce 35S Wx^a, 35S Wx^b was mutagenized using one of two mutagenic antisense primers, each of which creates a point mutation of T to G at the 5'-splice site of intron 1. A 526-bp SalI-NheI fragment of the PCR product was used to replace the corresponding fragment of 35S Wx^b. 35S Wx^b Stop and 35S Wx^a Stop were produced from 35S Wx^b and 35S Wx^a, respectively, by PCR, with one of two mutagenic antisense primers, each of which creates a nonsense mutation (TGG to TGA in exon 7). The 800-bp BamHI fragments of the PCR products were used to replace the corresponding fragments of 35S Wx^b and 35S Wx^a.

Transformation of Rice Protoplasts

Protoplasts (5 × 10⁶) were prepared from the rice Oc suspension cultures, mixed with 20 µg of plasmid DNA, and electroporated using a Gene Pulser (Bio-Rad Laboratories, Hercules, CA). After overnight incubation at 30°C, RNA was isolated from protoplasts for competitive RT-PCR or RNA decay measurements.

Competitive RT-PCR Analysis

The method for quantifying spliced waxy transcripts was as described previously (Isshiki et al., 2000). A competitor DNA for quantifying unspliced waxy transcripts was constructed by deleting a 61-bp DNA fragment from two DraI sites in intron 1 of Wx^a genomic DNA (exon 1-2) cloned into a pGEM-T vector. Serial dilutions of the competitor RNA transcribed in vitro were co-amplified with 500 ng of total RNA using an mRNA Selective PCR Kit (Takara, Kyoto). PCR cycle conditions were followed by 25 cycles (for endosperm RNA) or 40 cycles (for transformed Oc protoplast RNA) of 85°C for 1 min, 55°C for 1 min, and 72°C for 1 min with primers 5'-AACGGCCAGGATATTTATTGTG-3' and 5'-GAGAGCCGACATGGTGGTTG-3'. After PCR reactions, amplified DNA was electrophoresed in 2% (w/v) agarose gels and visualized by ethidium bromide staining. Quantitation of the amount of amplified fragments was performed as described previously (Gilliland et al., 1990).

RNA Decay Measurement

RNA decay measurements were performed with transformed Oc protoplasts. Actinomycin D (Sigma, St. Louis) was added to the cultures to a final concentration of 100 µg mL¹, and protoplasts were removed from the culture for 5, 10, and 20 min after actinomycin D addition. Each sample was immediately sedimented at 18,500g for 30 s and frozen in liquid nitrogen. Samples were analyzed by quantitative RT-PCR as described above.