| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online March 23, 2007; 10.1104/pp.107.096214 Plant Physiology 144:821-835 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Maize Zmsmu2 Gene Encodes a Putative RNA-Splicing Factor That Affects Protein Synthesis and RNA Processing during Endosperm Development1,[W],[OA]Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (T.C., C.S.K., H.N.N., B.A.L.); and Pioneer Hi-Bred International, a DuPont Company, Johnston, Iowa 50131–1004 (R.B.M.)
We characterized two maize (Zea mays) mutants, zmsmu2-1 and zmsmu2-3, that result from insertion of a Mutator (Mu) transposable element in the first exon of a gene homologous to the nematode gene, smu-2, which is involved in RNA splicing. In addition to having a starchy endosperm with reduced levels of zein storage proteins, homozygous zmsmu2-1 mutants manifest a number of phenotypes, including defective meristem development. The zmsmu2 mutants have poor seedling viability and surviving plants are sterile. The gene encoding ZmSMU2 is expressed in the endosperm, embryo, and shoot apex, which explains the pleiotropic nature of the mutation. We found that proper expression of Zmsmu2 is required for efficient ribosomal RNA processing, ribosome biogenesis, and protein synthesis in developing endosperm. Based on the pleiotropic nature of the mutations and the known function of animal Zmsmu2 homologs, we propose a possible role for ZmSMU2 in the development of maize endosperm, as well as a mechanism by which misregulation of zmsmu2 causes the mutant phenotypes.
The texture of maize (Zea mays) endosperm is an important quality trait, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. However, factors contributing to texture, i.e. hardness and vitreousness, are poorly understood (Mestres and Matencio, 1996  ; Chandrashekar and Mazhar, 1999). Kernel texture is partially related to the formation of a vitreous, or glassy, endosperm, which is influenced by the protein content of the seed and the conditions of the kernel during desiccation and storage. There appears to be a causal relationship between kernel hardness and the formation of zein protein bodies in the endosperm, because mutations that affect protein body size and the organization of zein proteins within them result in soft, starchy endosperms that cause an opaque kernel phenotype (Holding and Larkins, 2006). For example, Opaque2 (O2) encodes a transcription factor that regulates genes expressed in the endosperm, in particular those encoding 22-kD  -zeins (Schmidt et al., 1990). Protein bodies in o2 endosperm are smaller than wild type, and this has generally been considered to be the basis for the opaque kernel phenotype (Geetha et al., 1991; Segal et al., 2003). Several opaque mutants, e.g. floury2 (fl2) and Defective endosperm B30 (De-B30; Coleman et al., 1997; Kim et al., 2004a), are caused by defective signal peptides in -zein proteins that disrupt protein body assembly (Zhang and Boston, 1992), leading to increased expression of genes involved in the unfolded protein response (Hunter et al., 2002). These observations are consistent with the hypothesis that the size, number, and structure of zein protein bodies influence the texture and vitreous phenotype of the mature endosperm (Coleman and Larkins, 1999).
Although studies of the o2, fl2, and De-B30 mutants make it clear that zein proteins influence endosperm texture, other opaque mutants suggest there must be additional factors that contribute to this phenotype. For example, the O1 mutation (Nelson et al., 1965
To facilitate the identification and cloning of additional mutant genes that cause an opaque kernel phenotype, we screened a large population of Mutator (Mu)-tagged maize mutants created as part of Pioneer Hi-Bred's Trait Utility System for Corn (TUSC) collection (Bensen et al., 1995
Identification of mto38 and a Candidate Gene Responsible for the Opaque Kernel Phenotype
The mto38 mutant was discovered during a screen of Pioneer Hi-Bred's TUSC mutant collection (Bensen et al., 1995 During the backcrossing process, the mutant phenotype was monitored using a light box to screen for opaque kernels in segregating ears. Approximately 25% of the progeny from an F2 ear segregating for the mto38 mutation manifested an opaque kernel phenotype (Fig. 1A ), suggesting the mutation is due to a recessive allele. SDS-PAGE analysis of the zein and nonzein protein fractions from endosperms of these kernels revealed a marked reduction of the major zein storage proteins in the mutant (Fig. 1B, compare lanes 1 and 2) and quantitative differences in some nonzein proteins (Fig. 1B, compare lanes 3 and 4).
The mto38 kernels germinated poorly and rarely survived in the field. When the seeds were placed on Murashige and Skoog medium (Murashige and Skoog, 1962 ), their rate of germination improved, but the seedlings still had poor viability. Surviving plants were shorter than wild type and developed a sterile ear and a short, poorly branched sterile tassel. Because of the sterility, we were unable to propagate homozygous mto38 mutants; consequently, we maintained the mutant allele in heterozygotes. We frequently observed an unusual proliferation of leaves in homozygous mto38 seedlings (Fig. 2, A and B ), suggesting that meristem development was also affected. To investigate this, developing F2 kernels from a self-pollinated +/mto38 ear were harvested 16 d after pollination (DAP) and the endosperm and embryo dissected (note that embryos were genotyped by PCR after the nature of the Mu insertion was known). By 16 DAP, wild-type embryos formed five to seven leaves and had a small, distinct shoot apex (Fig. 2C). In contrast, mto38 embryos were smaller, and the apical meristem produced fewer leaves, as though development was delayed (Fig. 2, D–F). Some mto38 embryos had two shoot (Fig. 2D) and two root apices (Fig. 2E), while others possessed a single shoot and root apex (Fig. 2F). Thus, it appeared the gene responsible for mto38 is required for proper regulation of meristematic activity, and this may explain the low rate of germination and poor viability of the mutant seedlings.
To identify the Mu insertion responsible for the mto38 mutant, we used a thermal asymmetric interlaced PCR (TAIL-PCR) approach (Liu et al., 1995 ). DNA was extracted from 70 homozygous wild-type and 70 homozygous mto38 seedlings, and two sequential PCRs were carried out to amplify target DNA sequences with an arbitrary degenerate primer and nested Mu-specific primers that correspond to the Mu-terminal inverted repeat (MuTIR; Settles et al., 2004; see "Materials and Methods"). Upon gel electrophoretic separation of the products, we identified a DNA band of approximately 150 bp that was present in all the homozygous mto38 mutants (Fig. 3A
, lanes 1–6) but not homozygous wild-type seedlings (Fig. 3A, lanes 13–16). We also analyzed a few seedlings from nongenotyped vitreous F2 kernels (Fig. 3A, lanes 7–12). As predicted from the recessive nature of the mto38 allele, the 150-bp band was detected in some of these plants (Fig. 3A, lanes 8, 9, and 10), indicating they were probably heterozygous for the Mu insertion.
DNA sequence analysis of the 150-bp fragment revealed that approximately 90 bp corresponded to the MuTIR, while 60 bp were non-Mu DNA. To further characterize the Mu-associated DNA sequence, we designed two gene-specific primers, F1 and F2 (Fig. 3C; primers represented by arrowheads) to amplify the corresponding 5' and 3' ends (5'-RACE and 3'-RACE). While no product was obtained with the 5'-RACE, the 3'-RACE produced a cDNA containing a 1,698-bp open reading frame; the 5' end of this sequence was identical with the 60-bp non-Mu DNA sequence. Using this cDNA sequence, we designed a downstream gene-specific primer, R6, so we could easily genotype F2 individuals for the wild-type and mutant alleles. For genotyping the locus, PCR primers R6 and MuTir6 (Fig. 3C, gray arrowheads) were used to detect the mutant allele, while primers F3 and R6 were used to amplify the wild-type allele. With the F3-R6 primer pair, only the W64A+ but not the mto38 mutant allele could be amplified by PCR (Fig. 3B, lanes with a plus sign). Genotyping plants from vitreous and opaque kernels showed that all the F2 seedlings with an opaque phenotype (n = 45) were homozygous for the Mu-tagged fragment, while those from vitreous kernels (n = 136) were either homozygous wild type or heterozygous. This result confirmed that the Mu-tagged sequence was tightly linked with the opaque kernel phenotype. Nucleotide sequence analysis showed the cDNA encoded a hydrophilic protein consisting of 565 amino acids (Fig. 4 ). BLASTX comparison of the amino acid sequence against the National Center for Biotechnology Information protein database revealed a high degree of identity with RED-related proteins, which are conserved in higher eukaryotes. These proteins were named based on the RED domain, which contains several repeats of either Arg and Glu (RE repeats) or Arg and Asp (RD repeats). However, many of the homologous proteins, including all the plant accessions, do not contain distinct RE or RD repeats. Two highly conserved regions in the N and C termini were identified in the sequence alignment of the homologous proteins (Fig. 4), although no function has been assigned to these regions. We did not identify any of the other conserved domains, including those for binding to nucleic acids.
Among RED proteins, the human and nematode homologs are the best characterized. The human RED protein was identified in several independent proteomic studies where components of human spliceosomes were isolated (Neubauer et al., 1998 ; Makarov et al., 2002; Rappsilber et al., 2002; Zhou et al., 2002). The nematode homolog, smu-2, was isolated from a screen for genetic suppressors of the mec-8; unc-52 double mutant (Lundquist and Herman, 1994). SMU-2 plays a role in pre-mRNA splicing, because the smu-2 mutant accumulates unc-52 transcripts that lack exon 17, which contains a premature stop codon, thereby preventing the mec-8; unc-52 double mutant from being lethal (Spartz et al., 2004). The gene product of another suppressor, smu-1, physically interacts with SMU-2, and a human protein homologous to SMU-1 is also found in spliceosomes (Jurica and Moore, 2003). Because of the high level of sequence identity among smu-2 homologs and the biochemical properties of animal smu-2 homologs, we designated the mto38 candidate gene as Zmsmu2. In the mto38/zmsmu2-1 mutant, the Mu element appeared to be inserted in the first exon, which encodes the putative 5' untranslated region (UTR) of the mRNA. To verify this, it was necessary to define the structure of the gene. Using primers derived from the Zmsmu2 cDNA sequence, we amplified a 6.5-kb W64A+ genomic DNA fragment. Comparison of its sequence with that of the cDNA revealed the gene contains 11 exons (Fig. 3C). Because the Mu insertion made it difficult to obtain additional 5' genomic DNA sequence from W64A+, we screened a B73+ bacterial artificial chromosome (BAC) library with a Zmsmu2 cDNA probe. A comparison of the 5' ends of the W64A+ and B73+ Zmsmu2 genes revealed a poor conservation of nucleotide sequence near the Mu insertion site in zmsmu2-1 (boxed sequence in Fig. 3D), which sharply contrasted with their nearly identical downstream exonic and intronic sequences. Several potential cis-acting elements for binding of sequence-specific transcription factors were identified in the 5' sequence preceding the coding region of Zmsmu2 (underlined sequences in Fig. 3D). Thus, there did not appear to be additional exon or intron sequences upstream of the Mu insertion site in the W64A+ Zmsmu2 gene.
Based on the tight linkage of the zmsmu2-1 allele with the opaque kernel phenotype, we hypothesized this gene is responsible for the pleiotropic effects of the mutation. To test this, we investigated whether the zmsmu2-1 mutant exhibits defects in the expression of the Zmsmu2 gene and whether additional mutant alleles of Zmsmu2 result in similar phenotypes. Because the Mu insertion occurs in the first exon of Zmsmu2, it is possible this alters expression of the gene (Lisch, 2002 To investigate the level of ZmSMU2 protein in wild-type and mutant endosperm, ZmSMU2 antibodies were prepared by injecting a rabbit with antigen prepared after enzymatic removal of glutathione-S-transferase (GST) from a recombinant GST::ZmSMU2 fusion protein. The resulting antiserum recognized the recombinant fusion protein but did not bind GST (Fig. 5B, lanes 1 and 2). Immunoblotting of W64A+ endosperm proteins with these antibodies detected three polypeptides (Fig. 5B, lane 3) with apparent sizes of approximately 85, 75, and 55 kD. To determine which of these bands corresponds to the ZmSMU2 protein, we took advantage of the cross-reactivity of these antibodies with the homologous Arabidopsis (Arabidopsis thaliana) protein, AtSMU2, and its fusion protein GST::AtSMU2 (Fig. 5B, lane 4). We identified two Arabidopsis mutants, atsmu2-1 and atsmu2-2 (T. Chung, C.S. Kim, D. Wang, R. Yadegari, and B.A. Larkins, unpublished data), in which a T-DNA is inserted in the eighth exon and the sixth intron, respectively, of the single-copy AtSMU2 gene. These appear to be knockout mutants, because we were unable to detect a full-length cDNA from their RT reaction products (data not shown). Immunoblotting of extract from Arabidopsis seedlings at 7 d after germination (DAG) also identified three protein bands (Fig. 5B, lane 5). Based on the absence of the 100-kD band in extracts of homozygous atsmu2-1 and atsmu2-2 mutants (Fig. 5B, lanes 6 and 7), we concluded that the band of approximately 100 kD most likely corresponds to the AtSMU2 protein. Because ZmSMU2 is predicted to be 20 amino acids shorter than AtSMU2 (see Fig. 4 and also compare lanes 2 and 4 in Fig. 5B), the 85-kD band in lane 3 must correspond to the ZmSMU2 polypeptide, even though its apparent size is significantly larger (85 versus 64 kD) than the predicted molecular weight. The slower migration of this protein during SDS-PAGE presumably results from an unusual secondary structure or some type of posttranslational modification. We did not determine the nature of the smaller (75 and 55 kD) proteins, but they could share an epitope in common with the 85-kD ZmSMU2 band or represent degradation products. The level of ZmSMU2 protein in homozygous wild-type endosperm (Fig. 5C, lane 1) appeared to be slightly lower than in the zmsmu2-1 mutant (Fig. 5C, compare lanes 1 and 3). Furthermore, SDS-PAGE of 16 DAP endosperm extract of heterozygous and homozygous zmsmu2 genotypes revealed ZmSMU2 proteins with slightly different mobility. The wild-type protein appeared to be slightly larger than that in the mutant (Fig. 5C, compare lanes 1 and 3), and heterozygous (+/zmsmu2-1) endosperm contained proteins of both sizes (Fig. 5C, lane 2). This codominant pattern of ZmSMU2 inheritance was observed in nearly all the heterozygous F2 endosperms examined, with rare exceptions showing a banding pattern like the wild type (data not shown). Immunoblotting of proteins from different maize tissues was done to determine the spatial expression of the Zmsmu2 gene. This analysis showed comparable levels of the protein in developing endosperm (Fig. 5D, lane 1), embryo (Fig. 5D, lane 2), and shoot apex (Fig. 5D, lane 3). Unfertilized ovules (Fig. 5D, lane 6) contained lower but detectable amounts of ZmSMU2 protein, while none was found in pollen (Fig. 5D, lane 7). Very weak immunodetection was obtained with proteins from developing tassels, roots, and leaves, even when larger amounts of proteins were analyzed (data not shown). These results indicate that the maize Zmsmu2 gene is most highly expressed in mitotic/developing tissues, although it could be expressed throughout the plant. In summary, the Mu insertion in the first exon of the Zmsmu2 gene alters the 5' UTR of the mRNA transcript and increases its level. The difference in Zmsmu2 RNA levels between the wild-type and mutant endosperm is positively correlated with the protein levels. Significantly, the ZmSMU2 protein in mutant endosperm has a slightly smaller apparent molecular weight.
If the zmsmu2-1 mutation creates the diverse phenotypes of the mto38 mutant, additional mutant alleles of this gene should do the same. To test this, a reverse genetic screen of Pioneer Hi-Bred's TUSC population was conducted to identify additional Mu insertions in the Zmsmu2 gene. We were able to identify several Mu insertions at the 5' end of the Zmsmu2 gene, but nowhere else, even though we tested PCR primers corresponding to multiple intron and exon sequences. One of the Mu-tagged mutants, zmsmu2-3, had a Mu insertion in the first exon, 266 bp before the start codon, which is 59 bp downstream of the zmsmu2-1 insertion site (see Fig. 3, C and D). The other Mu insertions, zmsmu2-2 and zmsmu2-4, occurred in the first and second introns, respectively. No mutant phenotypes were observed for zmsmu2-2 and zmsmu2-4, suggesting introns containing these Mu insertions are removed by splicing during transcription. To eliminate Mu elements and obtain a more uniform genetic background, we introgressed the zmsmu2-3 allele into W64A+ by two or three generations of backcrossing. Subsequently, F2 progeny from the ear of a self-pollinated heterozygous zmsmu2-3 plant was compared with those from a self-pollinated heterozygous zmsmu2-1 plant (Table I ). The genotype for each of the F2 progeny was determined by PCR, and the number of leaves was counted at 10 DAG as an index for growth and development. Notably, homozygous zmsmu2-3 mutant kernels often failed to germinate or exhibited delayed seedling growth when compared to homozygous wild-type and heterozygous zmsmu2-3 siblings. Poor germination and delayed seedling growth were also observed in homozygous zmsmu2-1 mutants (Table I). After germination, homozygous zmsmu2-3 plants grew slowly, similar to the zmsmu2-1 mutant. Tassels and ears of the homozygous zmsmu2-3 plants were either absent or poorly developed, as with zmsmu2-1. Thus, in terms of germination and seedling growth, the zmsmu2-3 mutation appeared to phenocopy zmsmu2-1.
To determine if the Mu insertion in the zmsmu2-3 allele altered its expression, as well as that of other genes, we analyzed RNAs and proteins in wild-type and homozygous zmsmu2-1 and zmsmu2-3 mutant endosperms at 16 DAP. Compared to homozygous and heterozygous wild-type endosperm, there was a reduction in the level of zmsmu2-3 RNA (Fig. 6A ) and protein (Fig. 6B) in the homozygous mutant. This contrasted with homozygous zmsmu2-1 endosperm, where the gene was more highly expressed than in wild type (compare Fig. 6, A and B; see also Fig. 5). SDS-PAGE analysis showed that the mobility of the ZmSMU2-3 protein, like that of zmsmu2-1, was altered relative to wild type (Fig. 6B). In both cases, the change in size appeared to be small, but the difference was reproducible. The nucleotide sequence of the zmsmu2-3 cDNA was only slightly different from the wild-type and zmsmu2-1 alleles as a result of substitutions of Glu-107 and Ala-233 with Asp-107 and Ser-233 (see asterisks in Fig. 4). However, these amino acid substitutions do not explain the change in mobility detected by SDS-PAGE, because the molecular weight predicted for the protein is increased only 2 D.
An opaque kernel phenotype was observed in kernels developed from some of the zmsmu2-3 backcross populations, but a starchy endosperm was not as penetrant in this mutant as in zmsmu2-1. Nevertheless, zmsmu2-3 resulted in a reduction in zein synthesis, particularly -zeins, as was true of developing zmsmu2-1 endosperm (Fig. 6C).
We observed about a 50% decrease in total RNA per gram fresh weight (FW) of developing zmsmu2-1 endosperm compared with wild type, in spite of only a slight difference in their mRNA levels (Table II ). Furthermore, transcript profiling of zmsmu2-1 endosperm indicated that genes encoding ribosomal proteins and many ribosome biogenesis factors, especially rRNA processing proteins, appeared to be up-regulated in the mutant (T. Chung, C.S. Kim, D. Wang, R. Yadegari, and B.A. Larkins, unpublished data). Consequently, we decided to assess rRNA processing in zmsmu2-1 endosperm.
Figure 7A illustrates the primary transcript of a maize rDNA gene. Although rRNA processing sites have not been defined in the primary transcript, nucleotide sequences for rDNA gene repeats and their rRNA components, 18S, 5.8S, and 25S rRNA, were available. Based on this information, we designed six probes for northern-blot analysis (Fig. 7A), which allowed a comparison of mature and unprocessed rRNAs between wild-type and zmsmu2-1 endosperm. An rRNA intermediate containing the 5.8S rRNA, internal transcribed spacer 2 (ITS2), and 25S rRNA accumulated at higher levels in zmsmu2-1 endosperm (Fig. 7B, band b in blots 4–6), suggesting that the mutation leads to defective rRNA processing at cleavage sites in ITS2. An effect on rRNA processing in the mutant was also shown by the accumulation of unprocessed primary rRNA transcript (Fig. 7B, band a in all blots). Based on these observations, we concluded that the reduced yield of rRNA and the induction of ribosome biogenesis genes in zmsmu2-1 endosperm resulted from defective rRNA processing.
To test if the zmsmu2-3 mutant also manifests defective rRNA processing, we performed a similar experiment with 16-DAP zmsmu2-3 endosperm. RNA-blot analysis revealed that rRNA processing in zmsmu2-3 endosperm resulted in a slightly higher level of unprocessed rRNA than in wild-type or heterozygous sibling endosperms (Fig. 7C). To investigate whether the zmsmu2-1 mutation affected the translational efficiency of mRNAs, polysomes from equal amounts of 16-DAP wild-type and mutant endosperms were analyzed by Suc density gradient centrifugation. Using an ISCO 640 density gradient fractionator, the size distribution of polysomes was monitored by continuous UV absorbance as the Suc gradient was divided (top to bottom) into 0.8-mL fractions. Comparison of the wild-type and mutant polysome profiles provided additional evidence of a lower concentration of ribosomes in zmsmu2-1 endosperm (Fig. 8A ). There was an approximately 15% reduction in total ribosomal material (monosomes plus polysomes) in zmsmu2-1 endosperm, although this value was most likely underestimated, because some of the large polysomes in the wild-type sample pelleted to the bottom of the gradient. The highest absorbing polysome size-class in wild-type endosperm contained nine to 10 ribosomes, while that in zmsumu2-1 endosperm contained only four to five ribosomes per mRNA. This reduction in polysome size was observed in four independent pools of mutant endosperms, suggesting that the zmsum2-1 mutation not only results in fewer ribosomes per cell, but also that their translational activity is reduced.
An RT-PCR analysis was done to determine the distribution of selected RNAs among the polysome size classes. Based on the concentration of rRNA (Fig. 8B, 1 and 2) and mRNAs (Fig. 8B, 3–8  ), gradient fractions 6 to 8 had the highest amount of monosomes, while mRNAs in polysomes were recovered in fractions 9 to 13 (Fig. 8B). The highest concentration of ubiquitin conjugase (Ubc) mRNA, which was used as an internal control for semiquantitative RT-PCR, was found in fractions 10 and 11 of wild-type polysomes, compared with fractions 9 and 10 for polysomes from the zmsmu2-1 mutant. Evidence for less efficient mRNA translation was also found by the analysis of ribosomal protein S29 (Rps29) transcripts (Fig. 8B, 5 and 6). The total amount of Rps29 transcripts in monosome and polysome fractions was larger in the mutant. However, the peak concentration of mRNA in wild-type polysomes was in fractions 9 and 10, while in zmsmu2-1, the peak was in fractions 7 to 9, and a large portion of the RNA was in fractions 5 and 6, which corresponds to monosomes and, potentially, ribonucleoproteins. Similarly, the peak concentration for mRNA encoding one of the 22-kD -zeins was in fractions 12 and 13, while the corresponding mRNA in zmsmu2-1 peaked in fractions 10 and 11. In contrast, the Zmsmu2 RNA appeared to be similarly distributed in large polysomes in both wild-type and mutant endosperm (Fig. 8B, 7 and 8), although the level of transcripts was higher in the mutant. This result is consistent with previous analyses, which showed a higher level of zmsmu2-1 RNA and protein in the mutant endosperm (see Fig. 5C). To determine if altered rRNA processing in the mutant might have a functional significance, we hybridized the polysome-associated rRNA with the rRNA ITS2 probe. This analysis showed that more rRNA intermediates retaining the ITS2 sequence were found in the monosome fraction of the mutant than wild-type endosperm, while polysomes of the mutant contained very little of this intermediate (Fig. 8C). Thus, it is possible the presence of unprocessed rRNA contributes to the reduced translational efficiency of the ribosomes.
The opaque kernel mutant we identified as mto38 was found to result from a Mu insertion in the 5' UTR of a gene having a high degree of sequence identity with the RED proteins in mammals and C. elegans. The human RED protein is a component of spliceosomes (Neubauer et al., 1998 ; Makarov et al., 2002; Rappsilber et al., 2002; Zhou et al., 2002). In C. elegans, SMU-2 and a related protein, SMU-1, were shown to play an important role in splice site selection during alternative RNA splicing (Spartz et al., 2004). Consequently, we designated the maize mutant gene Zmsmu2. In maize, the zmsmu2 mutation has pleiotropic effects on gene expression that affect both endosperm and embryo development. The zmsmu2 mutation affects embryo morphogenesis with consequent effects on shoot and root meristem development, and the mutation negatively affects seedling viability, making it impossible to propagate homozygous mutant plants. Because we were most interested in the effects of the mutation on endosperm development, we did not pursue analysis of the mutation on gene expression during embryogenesis. Several lines of evidence support the conclusion that zmsmu2 is responsible for most if not all of the mutant phenotypes of mto38. First, we observed tight linkage between the Mu-tagged zmsmu2-1 allele and the opaque kernel phenotype (Fig. 3B). Second, through a reverse genetics screen, we identified additional Mu insertions in this gene, one of which, zmsmu2-3, manifested many of the same phenotypes as zmsmu2-1, including reduced germination and seedling viability and abnormal development of the shoot apical meristem, leading to proliferation of small leaves and reduced levels of zein synthesis during endosperm development. Like zmsmu2-1, the zmsmu2-3 mutation also resulted in inefficient rRNA processing in endosperm (Fig. 7C) as well as the up-regulation of ribosomal protein genes (T. Chung, C.S. Kim, D. Wang, R. Yadegari, and B.A. Larkins, unpublished data). Comparison of the Zmsmu2 genes in W64A+ and B73+, relative to Zmsmu2 cDNAs, indicated the zmsmu2-1 and zmsmu2-3 mutations are created by Mu insertions in the first exon of the gene, which encodes the 5' UTR of the mRNA. Initially, it was difficult to determine the correct start codon for Zmsmu2 transcripts, because the approximately 250-bp sequence corresponding to the proposed 5' UTR (see Fig. 3D) does not contain either a start or a stop codon. Based solely on the sequence analysis, we could not exclude the possibility of a start codon further upstream, i.e. additional exon/intron sequences. However, several lines of evidence suggest this is unlikely. First, when we searched the protein database using the 80 amino acid residues deduced from the 5' UTR, we were unable to find sequence similarity to any proteins; notably, they did not align with the N-terminal amino acid sequences of animal SMU-2 homologs. Second, all the plant SMU-2 homologs aligned very nicely at their N-terminal ends when the proposed initiation codon was used to deduce the ZmSMU2 amino acid sequence (Fig. 4). It is difficult to conceive that only the maize protein would have a longer N-terminal sequence. Third, we were unable to further extend the sequence of the 5' end of the wild-type TAIL-PCR product by 5' RACE. Finally, comparison of the W64A+ genomic sequence with that of B73+ showed that their nucleotide sequence identity quickly disappeared prior to the beginning of the 5' UTR. Because the nucleotide sequences of the W64A+ and B73+ alleles are identical in the first intron and subsequent sequences, this effectively argues against the likelihood there are exon or intron sequences further upstream of the proposed 5' UTR. Furthermore, we identified potential cis-elements for sequence-specific transcription factors in this region, implying it is part of the Zmsmu2 promoter sequence.
Previous studies have shown that Mu insertions in a genomic sequence corresponding to the 5' UTR of a transcript can cause changes in gene expression (Barkan and Martienssen, 1991 Our analysis of maize BAC clones showed that the Zmsmu2 gene maps in bin 10.01 of chromosome 10, although putative paralogs, represented by weaker hybridization signals, were found on chromosomes 3 and 8 (data not shown). Most of the maize ESTs encoding the ZmSMU2 protein are identical to the Zmsmu2 sequence, indicating that this gene encodes the predominant ZmSMU2 protein. The potential Zmsmu2 paralogous genes on chromosomes 3 and 8 could be nonfunctional copies, as a search of genomic databases showed that the rice (Oryza sativa) genome has only one functional copy of the OsSMU2 gene (Os11g04950) and two pseudogenes, Os12g04780 and Os12g04940, the latter of which could encode a truncated paralog of OsSMU2 (supported by an EST, CK041467.1). The zmsmu2-1 and zmsmu2-3 mutations create a change in the ZmSMU2 protein that leads to altered mobility during SDS-PAGE (Figs. 5C and 6B), although the mechanism is unclear. Because we were unable to identify any differences between the deduced primary amino acid sequences of the wild-type and ZmSMU2-1 mutant proteins, there are at least two plausible explanations for the altered mobility. First, variation in the 5' UTR of the zmsmu2-1 and zmsmu2-3 RNAs could affect usage of a downstream start codon, such as Met-13. The size differences of the proteins and their codominant expression pattern in heterozygous endosperm are consistent with this explanation. An alternative explanation is that the variation in sizes of the wild-type and mutant proteins results from some type of posttranslational modification. While we do not know the nature of this modification, phosphorylation is one possibility. Nevertheless, it is unclear what would bring about a change in the mutant protein's conformation that would lead to differential posttranslational modification.
We cannot pinpoint which aspect of zmsmu2 gene expression is responsible for the opaque kernel phenotype. The difference in ZmSMU2 protein level between the wild-type and zmsmu2 mutants could be responsible for mutant phenotypes. Many splicing regulators can affect processing of their target pre-mRNAs in a concentration-dependent manner (Smith and Valcarcel, 2000 Our data are consistent with the hypothesis that ZmSMU2 is a splicing factor. Among the evidence supporting this conclusion are the following observations: (1) mutations in zmsmu2 cause pleiotropic mutant phenotypes (Figs. 1 and 2); (2) the deduced amino acid sequence of ZmSMU2 is highly similar to a human spliceosomal protein and the nematode SMU-2, which is involved in alternative splicing (Fig. 4); and (3) ZmSMU2 showed a high level of tissue specificity, as is true of many Ser/Arg proteins (Fig. 5D). Furthermore, we identified differential pre-mRNA splicing events in zmsmu2 endosperm as well as protein interactions that imply a role for ZmSMU2 in pre-mRNA splicing (T. Chung, C.S. Kim, D. Wang, R. Yadegari, and B.A. Larkins, unpublished data).
zmsmu2-1 is the first plant mutant showing defective processing of nonorganellar rRNA (Fig. 7). Inefficient rRNA processing in this mutant is associated with a reduction in rRNA and, presumably, ribosomes, which could explain the global change in polysome sizes (Fig. 8, A and C). The mutant endosperm appears to accumulate increased levels of ribosomal protein transcripts that could be translated into ribosomal proteins (Fig. 8B). However, these proteins might not be assembled into functional ribosomes, because we observed a smaller pool of ribosomes in mutant than wild-type endosperm (Fig. 8A). The unassembled ribosomal proteins might be degraded, as occurs in yeast (Saccharomyces cerevisiae; Warner et al., 1985
We do not know how the zmsmu2-1 mutation causes defective rRNA processing in the endosperm. One explanation is that ZmSMU2 may participate in rRNA processing independently of its function in pre-mRNA splicing. This is a possibility, because yeast Prp43p was recently found to have dual functions in pre-mRNA splicing and ribosome biogenesis (Combs et al., 2006
Cloning of Maize zmsmu2-1 by Mu-TAIL PCR
To identify the Mu insertion responsible for the opaque kernel phenotype of mto38, genomic DNA was prepared from leaf tissue of 40 homozygous wild-type and 40 homozygous mto38 seedlings, and DNA sequences flanking the Mu elements were amplified by TAIL-PCR (Liu et al., 1995
Total RNA was isolated from homozygous W64A+ and zmsmu2-1 endosperms at 8, 10, 12, 14, 16, 18, and 20 DAP. A 3' RACE system kit (GIBCO BRL) was used to amplify the Zmsmu2 cDNA (GenBank accession no. EF460507), and the product was cloned into the pCR4-TOPO vector (Invitrogen) for DNA sequence analysis. Primary 3' RACE-PCR was carried out with the primer F1 (5'-caacagcggaacacgagggccaaatcg-3') based on the 5' sequence of the 150-bp fragment associated with the Mu insertion and the universal amplification primer that came with the RACE kit. Primary PCR reactions were performed according to the manufacturer's instructions. Aliquots (2 µL) from 100-fold dilutions of the primary PCR products were used directly for secondary 3' RACE-PCR reactions (20 µL). These were initiated by denaturing the cDNA at 94°C for 5 min, followed by 30 cycles of PCR as follows: 94°C, 30 s; 58°C, 30 s; 72°C, 1.5 min, using upstream primer F2 (5'-atcacttcgcctccgccctc-3') and the UAUP downstream primer in the RACE system kit. The final cycle was extended at 72°C for 5 min. The 1.6-kb cDNA obtained from this reaction was cloned into pCR4-TOPO and its nucleotide sequence subsequently determined.
Genotyping of the Zmsmu2 wild-type alleles was carried out by genomic PCR, using the upstream primer F3 (5'-acacgagggccaaatcgaaaaaatcactt-3') and the downstream primer R6 (5'-gcatcaacttctccttatagtagttcttc-3'), while genotyping for zmsmu2-1 and zmsmu2-3 alleles utilized the upstream primer MuTir6 and the downstream primer R6. Genomic DNA was denatured at 94°C for 5 min, followed by 35 cycles of PCR as follows: 94°C, 30 s; 54°C, 45 s; and 72°C, 45 s. The final cycle was extended at 72°C for 5 min. The PCR products were analyzed by 1.2% (w/v) agarose gel electrophoresis.
To PCR amplify the Zmsmu2 gene from W64A+, we designed the primers S1 (5'-tcccccgggggatcagccacgctgtttcttcgagct-3') and E1 (5'-ggaattcatgtcatcgaagaagaactactataag-3'). PCR conditions were as follows: 5 min at 94°C (first cycle); 30 s at 94°C, 30 s at 56°C, and 5 min at 72°C (35 cycles); and 5 min, 72°C (last cycle). The DNA products were inserted into pCR4-TOPO (Invitrogen) for sequencing (GenBank accession no. EF460506). To obtain B73+ BAC clones containing Zmsmu2, we screened a maize (Zea mays) genomic library ZMMBBb (http://www.genome.arizona.edu/orders/) with a radiolabeled probe from a 353-bp KpnI fragment corresponding to the 3' portion of the Zmsmu2 cDNA. Hybridization and identification of positive BAC clones were performed as described in http://www.genome.arizona.edu/information/protocols/addressnew.html. Of the 10 BAC clones with strongest hybridization signals, six clones, b0013K09, b0078C03, b0152G15, b0018M23, b0232J06, and b0098O15, were located in contig 392 of the maize fingerprint contig map (http://www.genome.arizona.edu/fpc/maize/). After the BAC clone, b0013K09, was digested with EcoRI, HindIII, or BamHI, the fragments were introduced into pBluescript. A radiolabeled genomic PCR product amplified with primers F3 and R6 was used for the secondary screen of the 5' UTR and promoter region of Zmsmu2 gene (GenBank accession no. EF460508).
To identify additional zmsmu2 alleles, approximately 42,000 F1 maize plants obtained from a cross with a Mu-active line were analyzed. DNA pools from this population were screened using the TUSC procedure at Pioneer Hi-Bred (Bensen et al., 1995
Embryos were dissected from 16-DAP kernels and fixed in 4% (v/v) formaldehyde in PHEM-dimethyl sulfoxide buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, 5% [v/v] dimethyl sulfoxide, pH 6.9) overnight at 4°C. They were then dehydrated in an ethanol series and infiltrated with Steedman's wax (Brown and Lemmon, 1995
Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. When RNA was extracted from developing endosperm, 50-mg samples were homogenized in SDS-LiCl buffer (50 mM Tris-Cl, pH 8.0, 150 mM LiCl, 5 mM EDTA, 1% SDS) and phenol-chloroform extracted, followed by conventional TRIzol extraction. After precipitation, RNA pellets were resuspended in 50 µL of ribonuclease-free water (1 µL/mg FW). For RT, 2.0 µg of total RNA and 0.5 µg of oligo(dT)12-18 were used in a 20-µL Superscript II reaction (Invitrogen). When RT was performed using RNA extracted from zmsmu2-1 endosperm, the same volume of RNA solution was used as wild type. This resulted in a more uniform amplification of Ubc control transcripts among the samples, because the yield of total RNA per gram FW was lower from mutant than wild-type endosperm, but the mRNA yield was only slightly reduced in the mutant. Reproducibility of the RT-PCR results was verified by three repetitions of independent experiments involving at least two biological replicates. Nucleotide sequences for the RT-PCR primers were as follows: ubc, ZmUBC_F1 (5'-aagatgcaggcatctagggcaagg-3') and ZmUBC_R1 (5'-aggctcttggcttggcacatgttc-3'); rps29, ZmrpS29_F1 (5'-atgggacactccaacgtgtggaac-3') and ZmrpS29_R2 (5'-ggttcgacatgctcagctagcata-3'); 22-kD
For northern-blot analysis of rRNA, probes were designed based on the nucleotide sequences of the maize rDNA gene (GenBank accession nos. K02202, U46605, and AJ309824). 32P-labeled probes were prepared by genomic PCR using W64A+ DNA as template and various pairs of primers as follows: 5' external transcribed spacer (ETS), ZmrDNA_5ETS_F1 (5'-tcggatgtggctacgcttgaaggc-3') and ZmrDNA_5ETS_R2 (5'-tagcacgtcctcgcagacgggcca-3'); 18S rRNA, ZmrDNA_18S_F3 (5'-cgttaacgaacgagacctcagcct-3') and ZmrDNA_18S_R4 (5'-ctgatgactcgcgcttactaggca-3'); ITS1, ZmrDNA_ITS1_F1 (5'-cagaccgcgaacgagtcacccgtg-3') and ZmrDNA_ITS1_R2 (5'-tcgattaaggtgtaaccgctgccc-3'); 5.8S rRNA, ZmrDNA_5.8S_F1 (5'-acgactctcggcaacggatatctc-3') and ZmrDNA_5.8S_R1 (5'-tgacgcccaggcagacgtgccctc-3'); ITS2, ZmrDNA_ITS2_F1 (5'-aagacactcccaacacccccccgc-3') and ZmrDNA_ITS2_R2 (5'-agggcaagctcggtcgctcgatgg-3'); and 25S rRNA, ZmrDNA_25S_F3 (5'-gaccgcgccgcgatagtaattcaa-3') and ZmrDNA_25S_R4 (5'-tcgtctgcaaaggattcagcacgc-3'). RNA was separated by agarose gel electrophoresis and transferred to nylon membrane as described in Sambrook and Russell (2001)
Polysome isolation was based on the methods described by Larkins (1985)
Zein and nonzein proteins from maize endosperm were prepared as described previously (Wallace et al., 1990
A Zmsmu2 cDNA containing the complete coding region was amplified by PCR with primers 38GEXE1 (5'-cggaattccatgtcatcgaagaagaactac-3') and 38GEXN1 (5'-agaatgcggccgctaatcagccacgctgtttcttcgag-3'). The DNA product was cloned into the EcoRI and NotI sites of pGEX4T-3 (GE Healthcare) to produce the fusion protein GST::ZmSMU2, and the plasmid was used to transform Escherichia coli strain BL21(DE3) codon+ (Stratagene). Bacterial cell extract in high-salt lysis buffer (10 mM phosphate buffer, pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride) containing 1% (v/v) Triton X-100 was incubated with glutathione-agarose beads (Sigma) at 4°C. After two washes with the high-salt lysis buffer and two washes with phosphate buffered saline (pH 7.3), the fusion protein on the beads was digested with thrombin (Sigma) to remove the GST tag. Following SDS-PAGE, 1 mg of ZmSMU2 protein was purified by gel excision and used as antigen for preparation of custom rabbit polyclonal antibodies (Strategic BioSolutions). Immunoblots were incubated with a 1:2,000 dilution of rabbit ZmSMU2 antiserum and then with 1:50,000 dilution of goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (Pierce).
The coding sequence of At2g26460 was amplified by RT-PCR using primers F + 60BspHI and R + 3924BamHI (5'-cgggatcccgtcaatgcttggatctcttagg-3') and the DNA product was introduced into pCR4blunt-TOPO (Invitrogen). The resulting plasmid, pCR4b-AtSMU2, was digested with EcoRI, reinserted into pGEX4T-3 (GE Healthcare), and used to transform the E. coli strain BL21(DE3) codon+ (Stratagene). The recombinant protein GST::AtSMU2 was purified by the same procedure as GST::ZmSMU2. The Arabidopsis (Arabidopsis thaliana) mutants atsmu2-1 (stock no. SALK_039202) and atsmu2-2 (stock no. WiscDsLox320H09) were obtained from the Arabidopsis Biological Resource Center.
Sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers EF460507, EF460506, and EF460508.
The following materials are available in the online version of this article.
We thank Dr. Rudolf Jung at Pioneer Hi-Bred for technical advice and assistance in gene expression analyses and colleagues in the Larkins lab for review and editing of the manuscript. We also thank Dr. Craig Coleman and Dr. Dwight Bostwick for their work on the TUSC screen that led to the identification of this mutant. Received January 24, 2007; accepted March 6, 2007; published March 23, 2007.
1 This work was supported by the Department of Energy (grant no. DE–96ER20242 to B.A.L.), by the National Science Foundation (grant no. 0077676), by the U.S. Department of Agriculture (grant no. CSREES 2004–00918), and by the Agricultural Plant Stress Research Center (Korea Science and Engineering Foundation, grant no. R112001092020080, partial salary support to C.S.K.). 
2 Present address: 425–G Henry Mall, Department of Genetics, University of Wisconsin, Madison, WI 53706.
3 Present address: Department of Plant Biotechnology and Agricultural Plant Stress Research Center, Chonnam National University, Kwangju 500–757, Korea. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Brian A. Larkins (larkins{at}ag.arizona.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.096214 * Corresponding author; e-mail larkins{at}ag.arizona.edu; fax 520–621–3692.
Allemand E, Gattoni R, Bourbon H-M, Stevenin J, Caceres JF, Soret J, Tazi J (2001) Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation. Mol Cell Biol 21: 1345–1359 Arthur KM, Vejlupkova Z, Meeley RB, Fowler JE (2003) Maize ROP2 GTPase provides a competitive advantage to the male gametophyte. Genetics 165: 2137–2151 Barkan A, Martienssen R (1991) Inactivation of maize transposon Mu suppresses a mutant phenotype by activating an outward-reading promoter near the end of Mu1. Proc Natl Acad Sci USA 88: 3502–3506 Bensen RJ, Johal GS, Crane VC, Tossberg JT, Schnable PS, Meeley RB, Briggs SP (1995) Cloning and characterization of the maize An1 gene. Plant Cell 7: 75–84[Abstract] Brown RC, Lemmon BE (1995) Methods in plant immunolight microscopy. Methods Cell Biol 49: 85–107[Web of Science][Medline] Chandrashekar A, Mazhar H (1999) The biochemical basis and implications of grain strength in sorghum and maize. J Cereal Sci 30: 193–207[CrossRef] Coleman C, Larkins BA (1999) The prolamins of maize. In PR Shewry, R Casey, eds, Seed Proteins. Kluwer Academic Publisher, Dordrecht, The Netherlands, pp 109–139 Coleman CE, Clore AM, Ranch JP, Higgins R, Lopes MA, Larkins BA (1997) Expression of a mutant Combs DJ, Nagel RJ, Ares M Jr, Stevens SW (2006) Prp43p is a DEAH-box spliceosome disassembly factor essential for ribosome biogenesis. Mol Cell Biol 26: 523–534 Geetha KB, Lending CR, Lopes MA, Wallace JC, Larkins BA (1991) Opaque-2 modifiers increase Holding DR, Larkins BA (2006) The development and importance of zein protein bodies in maize endosperm. Maydica 51: 243–254[Web of Science] Hunter BG, Beatty MK, Singletary GW, Hamaker BR, Dilkes BP, Larkins BA, Jung R (2002) Maize opaque endosperm mutations create extensive changes in patterns of gene expression. Plant Cell 14: 2591–2612 Jurica MS, Moore MJ (2003) Pre-mRNA splicing: awash in a sea of proteins. Mol Cell 12: 5–14[CrossRef][Web of Science][Medline] Kim CS, Hunter BG, Kraft J, Boston RS, Yans S, Jung R, Larkins BA (2004a) A defective signal peptide in a 19-kd Kim T-H, Kim B-H, Yahalom A, Chamovitz DA, von Arnim AG (2004b) Translational regulation via 5' mRNA leader sequences revealed by mutational analysis of the Arabidopsis translation initiation factor subunit eIF3h. Plant Cell 16: 3341–3356 Kraus ME, Lis JT (1994) The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development. Mol Cell Biol 14: 5360–5370 Larkins BA (1985) Polyribosomes. In HF Linskens, JF Jackson, eds, Modern Methods of Plant Analysis. Springer-Verlag, Berlin, pp 331–352 Leeds NB, Small EC, Hiley SL, Hughes TR, Staley JP (2006) The splicing factor Prp43p, a DEAH Box ATPase, functions in ribosome biogenesis. Mol Cell Biol 26: 513–522 Lisch D (2002) Mutator transposons. Trends Plant Sci 7: 498–504[CrossRef][Web of Science][Medline] Liu Y-G, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463[CrossRef][Web of Science][Medline] Longman D, Johnstone IL, Caceres JF (2000) Functional characterization of SR and SR-related genes in Caenorhabditis elegans. EMBO J 19: 1625–1637[CrossRef][Web of Science][Medline] Lundquist EA, Herman RK (1994) The mec-8 gene of Caenorhabditis elegans affects muscle and sensory neuron function and interacts with three other genes: unc-52, smu-1 and smu-2. Genetics 138: 83–101[Abstract] Makarov EM, Makarova OV, Urlaub H, Gentzel M, Will CL, Wilm M, Luhrmann R (2002) Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298: 2205–2208 Matlin AJ, Clark F, Smith CWJ (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6: 386–398[CrossRef][Web of Science][Medline] Meeley R, Briggs S (1995) Reverse genetics for maize. Maize Genet Coop Newslett 69: 67–82 Mestres C, Matencio F (1996) Biochemical basis of kernel milling characteristics and endosperm vitreousness of maize. J Cereal Sci 24: 283–290[CrossRef] Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497[CrossRef] Nelson OE, Mertz ET, Bates LS (1965) Second mutant gene affecting amino acid pattern of maize endosperm proteins. Science 150: 1469–1470 Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A, Mann M (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet 20: 46–50[CrossRef][Web of Science][Medline] Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12: 1231–1245 Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schmidt R, Burr F, Aukerman M, Burr B (1990) Maize regulatory gene Opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA. Proc Natl Acad Sci USA 87: 46–50 Segal G, Song R, Messing J (2003) A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 165: 387–397 Settles AM, Latshaw S, McCarty DR (2004) Molecular analysis of high-copy insertion sites in maize. Nucleic Acids Res 32: e54 Smith CWJ, Valcarcel J (2000) Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci 25: 381–388[CrossRef][Web of Science][Medline] Spartz AK, Herman RK, Shaw JE (2004) SMU-2 and SMU-1, Caenorhabditis elegans homologs of mammalian spliceosome-associated proteins RED and fSAP57, work together to affect splice site choice. Mol Cell Biol 24: 6811–6823 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680 Wallace JC, Lopes MA, Paiva E, Larkins BA (1990) New methods for extraction and quantitation of zeins reveal a high content of Warner JR, Mitra G, Schwindinger WF, Studeny M, Fried HM (1985) Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover. Mol Cell Biol 5: 1512–1521 Zhang F, Boston RS (1992) Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize. Protoplasma 171: 142–152[CrossRef][Web of Science] Zhou Z, Licklider LJ, Gygi SP, Reed R (2002) Comprehensive proteomic analysis of the human spliceosome. Nature 419: 182–185[CrossRef][Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
-zein synthesis and alter its spatial distribution in maize endosperm. Plant Cell 3: 1207–1219
