Plant Cell, Vol. 10, 1453-1464, September 1998, Copyright © 1998, American Society of Plant Physiologists
Temperature-Sensitive Splicing in the Floral Homeotic Mutant apetala3-1
Robert W. M. Sablowskia and
Elliot M. Meyerowitza
a Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125
Correspondence to:
Elliot M. Meyerowitz, meyerow{at}cco.caltech.edu (E-mail), 626-449-0756 (fax).
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ABSTRACT |
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The floral homeotic gene APETALA3 (AP3) is required for stamen and petal development in Arabidopsis. The previously described ap3-1 allele is temperature sensitive and carries a missense mutation near a 5' splice site. The missense mutation lies within a domain of the AP3 protein that is thought to be important for protein–protein interactions, which suggests that temperature sensitivity of ap3-1 could reflect an unstable interaction with cofactors. Here, we show instead that the ap3-1 mutation causes a temperature-dependent splicing defect and that temperature sensitivity is not a property of the protein products of ap3-1 but of RNA processing, possibly because of unstable base pairing between the transcript and small nuclear RNAs. The unexpected defect of the ap3-1 mutant offers unique opportunities for genetic and molecular studies of splice site recognition in plants.
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INTRODUCTION |
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Temperature-sensitive mutants have been important experimental tools ever since the classic experiments of Horowitz and Leupold 1951 
, which helped to establish the one gene–one enzyme paradigm by using temperature-sensitive mutants of Neurospora and Escherichia coli. Based on the characterization of many temperature-sensitive mutants in different organisms, sensitivity to temperature is frequently a property of the mutant protein product, which at the nonpermissive temperature loses its active conformation or the ability to interact functionally with other proteins (e.g., Folkmanis et al. 1976 ; Zachgo et al. 1995 ).
In plants, temperature-sensitive mutants have been useful in studying the function of floral homeotic genes (Bowman et al. 1989 ; Zachgo et al. 1995 ), which specify the identity of floral organs. There are three classes of floral homeotic genes, designated A, B, and C (Bowman et al. 1991 ; Coen and Meyerowitz 1991 ). Class A is active in the first two floral whorls, B is active in whorls 2 and 3, and C functions in whorls 3 and 4. Thus, each whorl expresses a distinct combination of homeotic activities: class A alone specifies sepals, A and B together cause petal formation, B and C combined specify stamens, and C alone directs carpel formation. With minor variations, the ABC model has been validated in different species, including Arabidopsis, Antirrhinum, and petunia (Yanofsky 1995 ; reviewed in Riechmann and Meyerowitz 1997 ).
In Arabidopsis, function B is performed by a pair of homeotic genes: APETALA3 (AP3) and PISTILLATA (PI), whose orthologs in Antirrhinum are DEFICIENS (DEF ) and GLOBOSA (GLO), respectively (Bowman et al. 1989 ; Hill and Lord 1989 ; Sommer et al. 1990 ; Jack et al. 1992 ; Trobner et al. 1992 ; Goto and Meyerowitz 1994 ). The AP3, PI, DEF, and GLO proteins belong to the MADS box family of transcription factors and bind DNA in vitro only as AP3/PI and DEF/GLO heterodimers, which may explain why mutations in either class B gene cause very similar phenotypes and why the ectopic B function requires combined expression of both AP3 and PI (Jack et al. 1994 ; Krizek and Meyerowitz 1996 ). In addition, B function genes autoregulate positively: if the function of one B function gene is lost, RNA accumulation for both partner genes (AP3/PI or DEF/GLO) is not maintained (Jack et al. 1992 ; Schwarz-Sommer et al. 1992 ; Goto and Meyerowitz 1994 ). Furthermore, in transgenic plants expressing AP3 ubiquitously, although AP3 mRNA accumulates ectopically, the AP3 protein is absent if PI is not expressed, presumably because the heterodimer is the only stable form of these proteins (Jack et al. 1994 ; Krizek and Meyerowitz 1996 ).
Temperature-sensitive mutants have been isolated for AP3 (ap3-1 allele) and DEF (def-101 allele) (Bowman et al. 1989 ; Schwarz-Sommer et al. 1992 ). In both cases, mutant plants grown at low temperature produce nearly wild-type flowers, but growth at the nonpermissive temperature causes the second-whorl organs to develop as sepals (only A function present) and stamens to be replaced by carpelloid organs (only C function left in the third whorl). Such conditional mutants have been useful to verify that B homeotic function is required throughout organ development and to establish the stage in organ development up to which organ identity can be redirected by activation of a different homeotic function (Bowman et al. 1989 ; Zachgo et al. 1995 ).
DEF-101 has a lysine residue deleted at position 93 within a conserved domain of plant MADS proteins called the K box (due to its similarity to the coiled-coil segment of keratin), which potentially forms amphipathic 
helices (Ma et al. 1991 ). Based on the temperature-sensitive defect of def-101, it has been proposed that the K box participates in protein–protein interactions (Davies and Schwarz-Sommer 1994 ). Accordingly, dimerization of DEF-101 with GLO has been shown to be temperature sensitive in vitro, suggesting that unstable interaction with GLO is the basis for the temperature-dependent defect of DEF-101 in vivo (Zachgo et al. 1995 ). ap3-1 carries a missense mutation, resulting in a change from lysine to methionine at position 153 of the AP3 protein (Jack et al. 1992 ). Because Lys-153 lies within the K box of AP3, the temperature-sensitive phenotype of ap3-1 also has been considered consistent with a function of the K box in protein–protein interactions (Davies and Schwarz-Sommer 1994 ; Zachgo et al. 1995 ).
Here, we show that the temperature-dependent phenotype of ap3-1 mutants is not due to loss of activity of the AP3-1 protein or to disruption of interaction with cofactors at the nonpermissive temperature but is caused instead by a temperature-sensitive splicing defect. The altered nucleotide in the ap3-1 transcript is expected to interact with small nuclear RNAs (snRNAs) during splicing, suggesting that temperature sensitivity may arise from unstable RNA–RNA interactions. The splicing pattern of ap3-1 suggests that the defect occurs at an early splicing step, when intron/exon boundaries are defined, and is consistent with the hypothesis that splice site choice in plants is influenced by interactions across exons (exon definition; Berget 1995 ).
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RESULTS |
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The ap3-1 Mutant Has a Temperature-Dependent Splicing Defect
Figure 1 shows that ap3-1 flowers grown at 16°C have nearly normal petals and stamens, but in contrast to the wild type, ap3-1 grown at 28°C develops sepaloid organs in the second whorl and carpelloid organs instead of stamens (Bowman et al. 1989 ). In ap3-1, the penultimate nucleotide of the AP3 exon 5 is changed from A to U (Jack et al. 1992 ). As shown in Figure 2A, the mutation not only causes an amino acid change in the predicted AP3 protein but also weakens the match between the 5' splice site of intron 5 and the consensus plant 5' splice site (Simpson and Filipowicz 1996 ). Accordingly, splice site prediction using a neural network method (Hebsgaard et al. 1996 ; NetPlantGene server, http://www.cbs.dtu.dk/NetPlantGene.html) failed to recognize the mutated ap3-1 splice site, whereas all wild-type AP3 splice sites were predicted correctly (data not shown). The mutated 5' splice site raised the possibility of a splicing defect in ap3-1. To test this hypothesis, we used reverse transcription–polymerase chain reaction (RT-PCR) to compare splicing of introns 4, 5, and 6 in wild-type and ap3-1 flowers.
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Figure 1.
Phenotypes of Wild-Type and ap3-1 Flowers Grown at 16 or 28°C.
See also Bowman et al. 1989 ; wt, wild type.
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Figure 2.
Temperature-Sensitive Splicing Defect of ap3-1.
(A) Structure of the AP3 gene and ap3-1 mutation. Exons are represented by boxes and are numbered e1 to e7; angled lines indicate introns and are numbered i1 to i6. Arrows represent the primers used to generate the RT-PCR products shown in (B). Exon 5, which contains the ap3-1 mutation, is black. The asterisk indicates where the mutation is located. Sequences near the 5' splice site of intron 5 are shown for wild-type AP3 and for ap3-1. Slashes indicate the exon–intron boundary, and vertical lines indicate complementarity to the 3' sequence of the U1 snRNA. The plant consensus 5' splice site is also shown (Solymosy and Pollak 1993 ). wt, wild type.
(B) RT-PCR products from RNA extracted from wild-type or ap3-1 flowers grown at the indicated temperatures (four independent samples for each treatment). The clusters of multiple bands are probably caused by addition of A residues to the end of PCR products by Taq polymerase; cloning of the PCR products corresponding to each cluster of bands did not reveal heterogeneity of sequences between the primers. To the right, numbered boxes represent the exons included in the RT-PCR products; arrows represent the PCR primers.
(C) Partial sequences of the ap3-1 RT-PCR products shown in (B). Nucleotides (nt) flanking the sequences shown here were identical to those of the wild type. The ap3-1 mutation and the corresponding missense mutation in AP3–Met-153 are highlighted; the box contains the exon 5 sequence (which is missing from the mRNA encoding AP3 e5) and corresponding amino acids (aa).
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Figure 2B shows that RT-PCR of the AP3 mRNA sequence between the end of exon 4 and the beginning of exon 7 revealed the predicted 213-bp product in wild-type plants, both at 16 and 28°C. In contrast, two RT-PCR products were detected by using RNA from ap3-1 plants. The larger product corresponded to correctly spliced ap3-1 mRNA, encoding the predicted mutant protein with the missense mutation (AP3–Met-153; Figure 2C). The shorter product (171 bp) corresponded to aberrantly spliced ap3-1 transcript: exon 4 was joined directly to exon 6. The resulting protein product (AP3e5; Figure 2C) would have amino acids 139 to 152 deleted, with the remaining C-terminal sequences fused in frame to amino acid 138. Splicing of the ap3-1 transcript was affected by temperature: in plants grown at 16°C, both splicing products accumulated to similar levels; at 28°C, the product lacking exon 5 corresponded to 80% of the total ap3-1 mRNA (Figure 2B). Comparable results were obtained if RT was primed with poly(T), as shown in Figure 2B, or if the requirement for polyadenylation was bypassed by using a primer that annealed within exon 7 of AP3 (data not shown). In flowers homozygous for the complete loss-of-function ap3-3 mutation, splicing was normal, indicating that correct splicing of the AP3 transcript does not require AP3/PI activity (data not shown).
The results described above revealed that splicing of the ap3-1 transcript is temperature sensitive. The temperature-dependent Ap3-1 phenotype could originate from this splicing defect, from temperature-sensitive function of either of the two predicted AP3-1 protein products, or from a combination of these defects.
Neither of the Alternative Protein Products of ap3-1 Is Temperature Sensitive
To separate the effect of temperature-sensitive splicing from a possible temperature-sensitive function of the AP3–Met-153 or AP3e5 proteins, transgenic plants were generated that expressed one or the other protein under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (Figure 3A and Figure 4A).
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Figure 3.
AP3–Met-153 Is Active and Insensitive to Temperature.
(A) Diagram of the 35S::AP3–Met-153 construct. Each numbered box represents an AP3 exon (e1 to e7); the black box indicates exon 5, which is excluded from AP3e5. The asterisk indicates the position of the Lys-to-Met missense mutation. CaMV 35S indicates the constitutive viral promoter. NOS ter indicates the transcriptional termination sequence from the nopaline synthase gene.
(B) Floral phenotypes of a representative 35S::AP3–Met-153; ap3-3 line grown at the indicated temperatures.
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Of eight independent 35S::AP3–Met-153 lines obtained, five had flowers with multiple, unfused carpelloid organs in the fourth whorl. In plants lacking endogenous AP3 activity (homozygous for ap3-3), in addition to causing unfused carpels, 35S::AP3–Met-153 restored stamen formation, although petal development was not rescued. The phenotype of a representative 35S::AP3 line in the ap3-3 background is shown in Figure 3B. Similar phenotypes (including the failure to rescue petal development in ap3-3 plants) were caused by expression of wild-type AP3 from the 35S promoter (35S::AP3; Jack et al. 1994 ; R.W.M. Sablowski and E.M. Meyerowitz, unpublished data). The phenotype of 35S::AP3–Met-153 plants was not affected by temperature (Figure 3B).
From 12 independent 35S::AP3e5 lines, six were indistinguishable from wild-type plants, and six had dominant loss of AP3 function, varying from green petals, to sepaloid organs in the second whorl, to a phenotype resembling that of the null ap3-3 allele (data not shown). These results suggest that AP3e5 might function as a dominant inhibitor of AP3 function, although this would not be easily reconciled with the fact that ap3-1 is recessive (Bowman et al. 1989 ). Alternatively, loss of AP3 function could be due to cosuppression (reviewed in Meyer and Saedler 1996 ). To test these hypotheses, expression of the AP3e5 protein in different transgenic lines was analyzed by immunoblotting. Expression of the transgene was tested in the absence of superimposed endogenous AP3, in homozygous ap3-3 plants (the ap3-3 mutation creates a premature stop codon 17 amino acids after the AP3 start codon; Jack et al. 1992 ). In heterozygous ap3-3/+ plants, the effect of AP3e5 on expression of the wild-type AP3 protein was examined. The genotypes of all plants were confirmed by PCR primer–introduced restriction analysis (data not shown; see Methods).
Figure 4B shows an immunoblot of extracts from 35S::AP3e5 flowers that were probed with the anti-AP3 antiserum. Three of the lines analyzed were phenotypically indistinguishable from the wild type (lines 1, 2, and 3). Line 1 did not express the transgene and had no effect on endogenous AP3. Lines 2 and 3 expressed AP3e5, which neither inhibited expression of wild-type AP3 (Figure 4B) nor restored AP3 activity in ap3-3 flowers, regardless of temperature (Figure 4C). The level of AP3e5 expressed in these lines was approximately half the level of AP3–Met-153 detected in a line showing clear rescue of AP3 function (see Figure 3 and Figure 6). Despite the lower levels of AP3e5 expression, if this protein had AP3 activity, it would likely still be detectable, because the 35S::AP3–Met-153 line used for comparison was relatively strong compared with the range of weaker phenotypes (partial rescue of stamen formation) seen in other 35S::AP3 and 35S::AP3–Met-153 lines (R.W.M. Sablowski and E.M. Meyerowitz, data not shown).
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Figure 5.
35S::ap3-1mg Is Temperature Sensitive.
(A) Diagram of the 35S::ap3-1mg and 35S::AP3mg constructs. Numbered boxes represent AP3 exons (e1 to e7); angled lines represent introns 4, 5, and 6 (i4 to i6); the asterisk indicates the position of the A-to-U mutation in ap3-1; the mutant exon 5 is represented by a black box in the ap3-1 minigene. CaMV 35S and NOS ter are as given in the legend to Figure 3A.
(B) Floral phenotypes of representative 35S::ap3-1mg and 35S::AP3mg lines both in the ap3-3 homozygous background and grown at the indicated temperatures.
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Figure 6.
Effect of Temperature on Accumulation of Wild-Type or Mutant AP3 Proteins.
An immunoblot was probed with the anti-AP3 antiserum, using protein samples from flowers with the genotypes indicated. Plants were grown at 16 or 28°C. The amount of total protein loaded on each lane was revealed by staining the blot with Ponceau S before blocking. All samples had similar amounts of proteins, except the wild-type (wt) sample at 28°C, which contained less total protein. The arrow indicates the band corresponding to the AP3, AP3e5, or AP3–Met-153 proteins. Bands indicated by the open circles are nonspecific, because they were detected in samples from ap3-3 flowers and also with the preimmune serum (data not shown).
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Two 35S::AP3e5 lines with loss of AP3 function were also tested (Figure 4B, lines 4 and 5). None expressed detectable AP3e5 protein, and both caused loss of expression of endogenous AP3, the degree of which correlated with the severity of their phenotypes (data not shown). Thus, interference with AP3 function in these lines was due to cosuppression. The reason the frequency of cosuppression was higher in 35S::AP3e5 than in 35S::AP3–Met-153 lines is not known.
In summary, AP3–Met-153 retained AP3 activity, whereas AP3e5 was inactive, and the activity of both proteins was unaffected by temperature. These results are compatible with the hypothesis that temperature-sensitive splicing is the origin of the temperature-dependent Ap3-1 phenotype.
Temperature Sensitivity of Transgenic ap3-1 Requires Intron Processing
35S::AP3–Met-153 and 35S::AP3e5 not only lacked the introns present in ap3-1 but they were also controlled by a different promoter. Although the specification of organ identity by the AP3–Met-153 protein was not affected by temperature, it could not be formally excluded that AP3–Met-153 could be temperature sensitive in its interaction with a cofactor specifically required for regulation of the AP3 promoter. To separate the effect of RNA processing from the activity of the AP3 promoter, we constructed an ap3-1 "minigene" (35S::ap3-1mg) containing the ap3-1 coding sequence interrupted by introns 4, 5, and 6 controlled by the CaMV 35S promoter, as represented in Figure 5A. A similar minigene lacking the ap3-1 mutation was also created (35S::AP3mg).
Figure 5B shows that the wild-type minigene (six independent 35S::AP3mg lines) completely rescued stamens and partially rescued petal formation, regardless of temperature, indicating that it contained all information required for correct processing of introns 4, 5, and 6. In previous work, expression of intronless AP3 from the CaMV 35S promoter failed to rescue petal formation in ap3-3 plants, possibly because of insufficient activity of the 35S promoter in the second whorl (Jack et al. 1994 ; Krizek and Meyerowitz 1996 ; R.W.M. Sablowski and E.M. Meyerowitz, unpublished data). The improved petal rescue by 35S::AP3mg suggested either that there are regulatory sequences within introns 4, 5, and 6, which are required for proper AP3 expression in the second whorl, or that intron processing stabilizes the AP3 mRNA, as observed for other plant transgenes (see Rose and Last 1997 ).
In four independent lines, 35S::ap3-1mg partially or completely rescued stamen formation in ap3-3 homozygous plants grown at 16°C, whereas at 28°C, the same lines developed carpelloid organs in the third whorl (Figure 5B). Thus, temperature sensitivity was conferred to the transgene by the requirement to process ap3-1 introns rather than by any aspect of promoter function. The 35S::ap3-1mg provided weaker AP3 activity than did the wild-type minigene, with incomplete stamen rescue and no rescue of petal formation.
Effect of Temperature on Accumulation of the AP3 Protein
The results described here suggest that in ap3-1 plants, sufficient levels of the active AP3–Met-153 would be produced at the permissive temperature, whereas at a higher temperature, the inactive AP3e5 would be the predominant product. To test this hypothesis, we examined accumulation of AP3-1 products at different temperatures by immunoblotting. Instead of showing a simple switch between the proteins produced, the immunoblot shown in Figure 6 reveals a marked reduction in the total levels of ap3-1 protein products at 28°C, whereas in wild-type plants there was little if any reduction (the lane corresponding to wild-type flowers at 28°C was underloaded; see the Figure 6 legend). This suggested that the effect of incorrect splicing of ap3-1 was amplified by interrupting the AP3/PI autoregulatory loop. Temperature also affected accumulation of the protein products from 35S::ap3-1mg lines but not from 35S::AP3mg (Figure 6), although the effect was less dramatic in 35S::ap3-1mg than in ap3-1 plants. Because transcriptional autoregulation was eliminated in these transgenes, at least part of the effect of temperature on accumulation of the ap3-1 protein products must be post-transcriptional. A direct effect of temperature on stability of AP3–Met-153 or AP3e5 is unlikely, because accumulation of these proteins in 35S::AP3e5 and 35S::AP– Met-153 was comparable at 16 and 28°C (Figure 6). Given that stability of the AP3 protein depends on active B function (Jack et al. 1994 ), the low levels of AP3e5 in 35S::ap3-1mg plants at 28°C may be an indirect effect of loss of B function at 28°C (see Discussion).
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DISCUSSION |
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Temperature-Sensitive Splicing
The results described here demonstrate that the temperature-dependent phenotype of ap3-1 is not a property of the AP3-1 protein products but reflects temperature-sensitive splicing. Several types of temperature-sensitive mutations affecting splicing have been characterized in different organisms. Some of these mutations affect proteins that control splicing events and therefore belong to the large group of temperature-sensitive mutations due to production of a defective protein product (Weidenhammer et al. 1996 ; Arenas and Abelson 1997 ; Ayadi et al. 1997 ; Staley and Guthrie 1998 ). Thermosensitive mutant snRNAs have also been described that presumably affect stability of RNA–RNA or RNA–protein interactions (Madhani et al. 1990 ; Alvarez et al. 1996 ; Maddock et al. 1996 ). The latter also have a broad effect on splicing. There are few reported cases of temperature-sensitive mutations that have a cis effect on splicing of a specific transcript. Apart from the example described here, similar cases have been reported in the murine retrovirus MuSVts110 (Touchman et al. 1995 ) and in the bimG gene of Aspergillus (Hughes et al. 1996 ). In both cases, the mutations are located near a 5' splice site, as in ap3-1. The likely basis for the temperature sensitivity of these 5' splice site mutants is unstable interaction with snRNAs known to form base pairs with sequences around the 5' splice site or defective interaction between the transcript and protein factors that participate in 5' splice site selection (see below).
The primary splicing defect, however, may not be sufficient to explain the marked loss of function of ap3-1 at the nonpermissive temperature. The low levels of ap3-1 products accumulated at 28°C suggest that the reduction in AP3 activity caused by the splicing defect may be amplified by failure to maintain the AP3/PI autoregulatory loop. Part of this effect is expected to be transcriptional, because there is evidence for activation of the AP3 promoter by AP3/PI (Jack et al. 1994 ; Krizek and Meyerowitz 1996 ). The reduced accumulation of protein products at 28°C in 35S::ap3-1mg plants, however, also indicates that post-transcriptional events were affected. Post-transcriptional regulation of AP3 has been revealed in 35S::AP3 plants: although the transgene caused ubiquitous accumulation of AP3 mRNA, immunolocalization showed that the AP3 protein accumulated only where PI was also expressed (Jack et al. 1994 ). The AP3/PI heterodimer appears to be the only stable form of the protein, although it is also possible that AP3/PI activity may be required for producing additional factors required for translation of the AP3 mRNA or stability of the AP3/PI protein.
Thus, the available data suggest that when ap3-1 plants are shifted to the nonpermissive temperature, the splicing defect causes a shift from production of active AP3–Met-153 to predominant production of mRNA encoding the inactive AP3e5; diminished AP3 activity causes reduced transcription of the AP3 and PI genes; lower levels of PI protein may in turn destabilize the ap3-1 protein products, causing B function to spiral down to even lower levels. Similarly, amplification of the primary splicing defect may occur in the other cis-acting temperature-sensitive mutants mentioned above. In the case of bimG in Aspergillus, the splicing product produced at the nonpermissive temperature is a dominant inhibitor of the wild-type product (Hughes et al. 1996 ), which should steepen the loss of function at the nonpermissive temperature. In the case of the MuSVts110 retrovirus, the effect of inefficient splicing at the nonpermissive temperature (Touchman et al. 1995 ) may be exacerbated by multiple cycles of viral replication.
The precedents set by ap3-1, bimG, and MuSVts110 raise the question of whether temperature-sensitive splicing due to a 5' splice site mutation is rare or whether its effects are usually not evident unless the primary defect is subsequently amplified. Apart from ap3-1, numerous other 5' splice site mutations have been described in plant genes (reviewed in Brown 1996 ), providing opportunities to test whether temperature-sensitive splicing occurs in any of these mutants.
Molecular Basis of the ap3-1 Splicing Defect
Splicing is catalyzed by a large ribonucleoprotein complex (spliceosome) that is formed by stepwise assembly of five small nuclear ribonucleoprotein particles (snRNPs U1, U2, U4/U6, and U5) on the pre-mRNA template (reviewed in Madhani and Guthrie 1994 ). In mammals and yeast, the 5' splice site is defined by interactions between the splice site sequence and U1, U5, and U6 snRNAs. The 5' end of the U1 snRNA is complementary to the consensus sequence around the 5' splice site and base pairs with it at an early stage of spliceosome assembly (Ruby and Abelson 1988 ). U5 later replaces U1 and interacts with the nucleotide at position -2 of the splice site before cleavage (Newman and Norman 1992 ; Wyatt et al. 1992 ); U5 has been proposed to hold the cleaved 5' exon in place for ligation to the 3' exon (Sontheimer and Steitz 1993 ). U6 is thought to align the 5' splice site and the branch point for the first splicing step and also participates in the ligation of the exons (Madhani and Guthrie 1994 ). In addition to snRNAs, interactions between proteins and pre-mRNA also participate in defining splice sites (Crispino et al. 1994 ; reviewed in Horowitz and Krainer 1995 ).
The consensus 5' splice site and relevant sequences of U1, U5, and U6 snRNAs are conserved in plants, mammals, and yeast (Solymosy and Pollak 1993 ), suggesting that the above-described interactions with pre-mRNA also occur in plants. If this is true, then the nucleotide that is mutated in ap3-1 would be expected to be part of the transcript sequences that interact with snRNAs. Although it cannot be excluded that the ap3-1 mutation could weaken an interaction between a protein and pre-mRNA, an attractive hypothesis for the origin of temperature sensitivity in ap3-1 would be that weaker RNA–RNA pairing would be disrupted at the higher temperature. The U6 snRNA contacts nucleotides +4 to +6 of the 5' splice site (Lesser and Guthrie 1993 ); therefore, it is unlikely that it would be affected directly in ap3-1. The U5 snRNA contacts nucleotide -2 (Wyatt et al. 1992 ), which is the position mutated in ap3-1, but this interaction is not base specific (Newman and Norman 1992 ; Wyatt et al. 1992 ; Sontheimer and Steitz 1993 ; Newman 1997 ). Because the mutation lies within the region expected to form canonical base pairs with the U1 snRNA, interaction with the latter is more likely to be affected in ap3-1 flowers. A precedent for temperature-sensitive base pairing between the U1 snRNA and 5' splice sites has been set by artificial mutant U1 snRNAs in Schizosaccharomyces pombe (Alvarez et al. 1996 ). In addition, the skipping of exon 5 in ap3-1 suggests a defect in communication between spliceosomes being assembled on adjacent introns (see below). Because the U1 snRNA has a role in communication between splice sites across exons (Kuo et al. 1991 ; Hoffman and Grabowski 1992 ), exon skipping in ap3-1 is also consistent with a defective interaction with the U1 snRNA.
The uridine present at position -2 in ap3-1 is also found in other plant 5' splice sites, including the 5' splice site of intron 6 of the AP3 gene (5'-CTG/GTAATA-3'). In the latter case, the better general match to the consensus 5' splice site may compensate for the mismatch at position -2. The degree of complementarity to the U1 snRNA, however, is not the only determinant of 5' splice site selection: protein cofactors and exon sequences are also involved in animal cells (Crispino et al. 1994 ; reviewed in Horowitz and Krainer 1995 ), yeast (Lockhart and Rymond 1994 ), and plants (Carle-Urioste et al. 1997 ; Egoavil et al. 1997 ). It seems likely that in ap3-1, base pairing between snRNAs and position -2 of the mutated 5' splice site becomes limiting because additional determinants of splice site recognition are suboptimal (see below).
Splice Site Definition in Plants
In mammalian cells, splice sites seem to be recognized in pairs that flank exons (exon definition; reviewed in Berget 1995 ). The small size of mammalian exons (average <300 nucleotides) relative to introns suggests why discriminating exon sequences would be more efficient than sequence-based recognition of introns. Part of the evidence supporting exon definition is the frequent observation that mutations in a 5' or 3' splice site cause skipping of the adjacent exon. Also consistent with exon definition is the observation that exon binding SR (serine–arginine repeat) proteins form a molecular bridge between the polypyrimidine tract binding factor U2AF65 and U1 snRNP bound to the 5' splice site of the downstream intron (reviewed in Reed 1996 ). Accurate selection of splice sites may be ensured by the cooperative action of multiple factors interacting weakly with sequence elements near splice sites and within exons (Reed 1996 ).
In plants, there is evidence that recognition of regions containing intron/exon boundaries is based on the transition between sequences where AU content is high (introns) and moderate (exons) (Goodall and Filipowicz 1989 ; Gniadowski et al. 1996 ). The exact 5' splice site is then chosen by scanning sequences upstream of these regions until appropriate complementarity to the U1 snRNA is found (McCullough et al. 1996 ; McCullough and Schuler 1997 ). 3' Splice sites are also thought to be found near a transition in AU content, by a combination of scanning and competition between potential splice sites (reviewed in Simpson and Filipowicz 1996 ). Such scanning mechanisms could operate independently in the definition of each intron or could be coordinated between introns by interaction across exons. It has been suggested that as in yeast, the small size of plant introns may not require exon definition (Berget 1995 ). On the other hand, exon skipping has been observed in several splice site mutants in Arabidopsis, suggesting that exon definition may occur in plants (Brown 1996 ). This possibility is also consistent with the existence of families of SR proteins in plants (Lazar et al. 1995 ; Lopato et al. 1996 ).
If introns were recognized as independent units and relied solely on outward scanning to define 5' splice sites, failure to recognize the defective 5' splice site of ap3-1 would lead to scanning for upstream cryptic 5' splice sites. In the absence of cryptic sites, intron retention would be expected. RT-PCR, however, failed to detect products with cryptic 5' splice sites or retention of intron 5 (also with primers covering the whole ap3-1 coding sequence; data not shown). In addition, a simple intron recognition scanning model does not provide a simple explanation for why a defect in the 5' splice site of intron 5 would be coupled with failure to recognize the 3' splice site of the upstream intron, leading to exon skipping. Although not proven by the available data, exon definition offers the most parsimonious explanation for the pattern of ap3-1 splicing. Skipping of exon 5 would occur because failure to recognize the weak 5' splice site of intron 5 in ap3-1 would abolish interactions across exon 5, which would be necessary for recognition of the 3' splice site of intron 4. One testable prediction of this model is that correct splicing of intron 4 would be restored by mutations that increased the strength of its 3' splice site. Strikingly, a second-site mutation, which suppresses the splicing defect in ap3-1, maps to intron 4, further indicating that interactions between introns 4 and 5 determine the efficiency of splicing of exon 5 and supporting the hypothesis that exon definition occurs in plants (Yi and Jack 1998 ).
Could Temperature-Sensitive Splicing Be Engineered?
If unstable RNA–RNA pairing between a transcript and snRNAs can be the basis for temperature-sensitive mutations, then it might be simpler to confer artificially temperature-sensitive expression to a gene by altering splice sites than it would be to engineer a temperature-sensitive protein product. As mentioned above, however, it is likely that splice site recognition results from the combined action of multiple factors (Reed 1996 ; Simpson and Filipowicz 1996 ). In the context of ap3-1, temperature-sensitive pairing to snRNA may be revealed because other factors for recognition of the 5' splice site of intron 5 are suboptimal. Attempts at engineering temperature-sensitive splicing will probably require a better understanding of these additional factors. Based on the evidence from other organisms, likely candidates would be exon-binding SR proteins and their corresponding exon enhancers and factors participating in 3' splice site recognition in the upstream intron (Reed 1996 ; McCullough and Schuler 1997 ).
If splicing of ap3-1 is precariously poised between success and failure, then small changes in any other factor affecting 5' splice site recognition could have a noticeable effect. This possibility, combined with the magnifying effect of the AP3 positive feedback regulation, suggests that additional factors affecting the 5' splice site in plants may be identified by screening for mutations that enhance or suppress the Ap3-1 phenotype. The results from a screen for suppressors of ap3-1 are reported by Yi and Jack 1998 .
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METHODS |
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Plant Growth
Seeds (Arabidopsis thaliana ecotype Landsberg erecta) were planted on a mixture of 4:3:2 soil–vermiculite–perlite, left at 4°C for 4 days, and grown at 16, 22, or 28°C under continuous fluorescent cool-white light (600 foot-candles) with weekly fertilization (Miracle-Gro 20-20-20; Stern's Miracle-Gro Products, Port Washington, NY).
Reverse Transcription–Polymerase Chain Reaction
Inflorescence tips (from the meristem to the first few mature flowers) were frozen in liquid nitrogen immediately after dissection. Tissues were ground in liquid nitrogen, and RNA was extracted with Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH), according to the manufacturer's instructions. DNase I treatment and cDNA synthesis from 300 ng of total RNA were as described previously (Reuber and Ausubel 1995 ), except that the first 10 min of reverse transcription (RT) was performed at 25°C and the primer used was 5'-TGCCGAAGCT12C-3'. Polymerase chain reaction (PCR) was performed in 25 µL of standard PCR buffer (Boehringer Mannheim) containing 1 µL of undiluted RT product, 2 µM each deoxynucleotide triphosphate, 1 µCi -33P-dATP (Amersham), and 100 ng each of primers 5'-GTCGTCTAGAGGATGAAATGGAAAACAC-3' and 5'-TTGGGATCCAAGAACTGAGTCGTAATC-3'. Reactions were started at 94°C by addition of 1 unit Taq polymerase (Boehringer Mannheim) followed by 35 cycles of 94°C for 30 sec, 50°C for 1 min, and 72°C for 2 min. Products were then analyzed on a 5% sequencing gel. The relative amounts of PCR products were determined by using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
For cloning, PCR reactions contained no -33P-dATP and 250 µM each deoxyribonucleotide phosphate. PCR products were digested with BamHI and XbaI and cloned into BamHI/XbaI–digested pBluescript KS- (Stratagene, La Jolla, CA). Sequencing of cloned products was performed with the Sequenase 2.0 sequencing kit (U.S. Biochemical Corp., Cleveland, OH).
Construction of Plasmids for Plant Transformation
All cloning techniques were standard (Sambrook et al. 1989 ). To create pAP3BX, the AP3 coding sequence from pF733 (Jack et al. 1992 ) was removed as an EcoRI-BanI fragment, filled in with the Klenow fragment of DNA polymerase I, and cloned into pBluescript KS- previously digested with SpeI and filled in with the Klenow fragment. pAP3–Met-153 and pAP3e5 were created by replacing the NruI-SpeI region of pAP3BX with NruI/SpeI–digested PCR fragments generated from ap3-1 cDNA, as described above. To construct pAP3mg and pap3-1mg, AP3 and ap3-1 genomic sequences were PCR amplified from genomic DNA from Arabidopsis (ecotype Landsberg erecta), using the same primers and conditions described for RT-PCR. The PCR products were digested with NruI/SpeI and again used to replace the corresponding fragment from pAP3BX. All pAP3BX derivatives described above were sequenced to confirm the absence of PCR errors. Plasmids for plant transformation were generated by removing the BamHI-XbaI fragments AP3–Met-153, AP3e5, AP3mg, and ap3-1mg from the corresponding pAP3BX derivatives and cloning into BamHI/XbaI–digested pCGN18 (Krizek and Meyerowitz 1996 ) to generate p35S::AP3–Met-153, p35S::AP3e5, p35S::AP3mg, and p35S::ap3-1mg, respectively.
Plant Transformation and Line Construction
p35S::AP3–Met-153, p35S::AP3e5, p35S::ap3-1mg, and p35S::AP3mg were transformed into the ASE strain of Agrobacterium tumefaciens. The transformed bacteria were then used to transform Arabidopsis (ecotype Landsberg erecta) by vacuum infiltration (Bechtold et al. 1993 ). Bleach-sterilized seeds were plated on Murashige and Skoog medium (Murashige and Skoog 1962 ) with 50 µg/mL kanamycin, stratified at 4°C for 4 days, and germinated for 10 days at 22°C under continuous 600 foot-candles of cool-white fluorescent light. Kanamycin-resistant plants were potted and grown as described above. Transgenic plants were manually cross-pollinated to combine the transgenes with the ap3-3 mutation.
PCR Genotyping
When necessary, the presence of the ap3-3 mutation was monitored by PCR from leaf pieces (Klimyuk et al. 1993 ) by using PCR primer introduced restriction analysis (Jacobson and Moscovits 1991 ) with the primers 5'-AGAGGATAGAGAACCAGACAAATCGA-3' and 5'-GT T-TAGAGAGATGGTGTACGTGG-3'. To avoid interference by AP3 sequences contained in the transgenes, the latter primer was designed to hybridize with sequences within intron 1 of AP3. PCR amplification was as described above for RT-PCR, with 250 µM each deoxyribonucleotide triphosphate. Eighteen microliters of the PCR product was added to 2 µL of solution containing Tris-HCl (400 mM; pH 7.5), MgCl2 (85 mM), and NaCl (500 mM). The mixture was then digested with ClaI and analyzed on a 3% agarose gel (of which 2% was low-melting-point agarose). The product amplified from ap3-3 was cut to 160 bp, whereas the wild-type 182-bp product lacked a ClaI site.
Immunoblotting
Inflorescence tips (from the meristem to the first few mature flowers) were frozen and ground in liquid nitrogen, thawed in 1 volume of sample buffer (2% SDS, 10% glycerol, 100 mM DT T, 60 mM Tris-HCl, and 0.001% bromphenol blue, pH 6.8), boiled for 2 min, and centrifuged (12,000g for 5 min). Ten microliters of the supernatant was then analyzed on a 15% SDS–polyacrylamide gel, blotted onto Hybond-C Extra (Amersham), and probed by using standard methods (Harlow and Lane 1988 ). The primary antibody was 1:2000 anti-AP3 rabbit serum (Jack et al. 1994 ), and the secondary antibody was 1:1000 donkey anti–rabbit Ig conjugated to horseradish peroxidase (Amersham). The blot was then developed with an enhanced chemiluminescence kit (Amersham).
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ACKNOWLEDGMENTS |
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We are grateful to Dr. Tom Jack for the pF733 plasmid and anti-AP3 antiserum and for communicating results before publication and to Drs. Gordon Simpson, Tracy Johnson, John Wagner, Tom Jack, Jennifer Fletcher, Prakash Kumar, Steve Jacobsen, Eva Ziegelhoffer, Carolyn Ohno, and Chiou-Fen Chuang for critical reading of the manuscript. R.W.M.S. was supported by a long-term fellowship (No. LT-12/95) from the Human Frontier Science Program, and work in E.M.M.'s laboratory is supported by a grant from the National Science Foundation (No. MCB-9603821).
Received April 20, 1998; accepted July 13, 1998.
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ABSTRACT
INTRODUCTION
