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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Molecular Characterization and Phylogeny of U2AF³⁵ Homologs in Plants^1,[W],[OA]

Department of Genetics, Development and Cell Biology (B.B.-W., V.B.) and Department of Statistics (V.B.), Iowa State University, Ames, Iowa 50010

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is an essential splicing factor with critical roles in recognition of the 3'-splice site. In animals, the U2AF small subunit (U2AF³⁵) can bind to the 3'-AG intron border and promote U2 small nuclear RNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit. Two copies of U2AF³⁵-encoding genes were identified in Arabidopsis (Arabidopsis thaliana; atU2AF³⁵a and atU2AF³⁵b). Both are expressed in all tissues inspected, with atU2AF³⁵a expressed at a higher level than atU2AF³⁵b in most tissues. Differences in the expression patterns of atU2AF³⁵a and atU2AF³⁵b in roots were revealed by a promoter::-glucuronidase assay, with atU2AF³⁵b expressed strongly in whole young roots and root tips and atU2AF³⁵a limited to root vascular regions. Altered expression levels of atU2AF³⁵a or atU2AF³⁵b cause pleiotropic phenotypes (including flowering time, leaf morphology, and flower and silique shape). Novel slicing isoforms were generated from FCA pre-mRNA by splicing of noncanonical introns in plants with altered expression levels of atU2AF³⁵. U2AF³⁵ homologs were also identified from maize (Zea mays) and other plants with large-scale expressed sequence tag projects. A C-terminal motif (named SERE) is highly conserved in all seed plant protein homologs, suggesting it may have an important function specific to higher plants.

Genes encoding U2AF³⁵ were designated U2AF1. Single to multiple copies of U2AF1 were identified in fission yeast (Schizosaccharomyces pombe; Wentz-Hunter and Potashkin, 1996), worm (Zorio and Blumenthal, 1999), fly (Rudner et al., 1996), fish (Tassone et al., 1999), chicken (Pacheco et al., 2004), and mammals (Zhang et al., 1992; Tupler et al., 2001; Shepard et al., 2002). U2AF³⁵ contains a pseudo-RNA recognition motif (RRM) flanked by two highly conserved C₈C₅C₃H zinc fingers (Kielkopf et al., 2001, 2004). The RRM and two zinc fingers are essential for U2AF³⁵ function (Webb and Wise, 2004). U2AF³⁵ also contains a C-terminal RS domain (Zhang et al., 1992), which was found to be dispensable in vivo (Rudner et al., 1998). In mammals, a recently duplicated copy of U2AF1 encodes a 26-kD protein (U2AF²⁶; Shepard et al., 2002). U2AF²⁶ lacks the C-terminal RS domain, but is still able to functionally substitute for U2AF³⁵ in splicing (Shepard et al., 2002). An alternatively spliced isoform of the U2AF1 pre-mRNA is conserved from fish to human (Pacheco et al., 2004). The encoded protein has seven amino acid differences in the RRM, but is still able to bind to U2AF⁶⁵ and promote U2AF splicing activity in vitro (Pacheco et al., 2004). Multiple copies and isoforms of U2AF1 may contribute to the fine-tuned control of pre-mRNA splicing in vertebrates (Pacheco et al., 2004). In yeast (Saccharomyces cerevisiae), however, no ortholog of U2AF³⁵ exists, although a functional ortholog of U2AF⁶⁵ was identified (Mud2p; Abovich et al., 1994). No SR proteins were found either, possibly because the conserved branch consensus sequence eliminates the requirements for these factors in yeast.

Mammalian introns can be classified into AG-dependent and AG-independent types (Reed, 1989). In the AG-dependent introns, the Py tract is short (weak) and the adjacent 3'-AG boundary is essential for splicing (Reed, 1989). The function of U2AF³⁵ is important in these introns because it stabilizes the binding of U2AF⁶⁵ with the weak Py tract (Zuo and Maniatis, 1996). AG-independent introns, however, have a long (strong) Py tract and the AG is not required for splicing (Reed, 1989). U2AF⁶⁵ alone is sufficient for recruiting the U2 snRNP to the branch-point sequence (Wu et al., 1999). Plant introns have neither conserved branch-point sequences nor a Py tract. Two U2AF⁶⁵ homologs isolated from wild tobacco (Nicotiana plumbaginifolia) can complement the in vitro splicing of adenovirus pre-mRNA in HeLa cell extracts depleted of U2AF factor (Domon et al., 1998). Previous results revealed that Arabidopsis (Arabidopsis thaliana) has three copies of genes encoding the U2AF large subunit and one possible pseudogene (Wang and Brendel, 2004). These results suggest that the mechanism of 3'-splice site (ss) recognition is conserved in plants. Because plant introns lack strong branch-point and Py-tract motifs, it is very likely that 3'-ss recognition in plants relies more on U2AF³⁵ to stabilize binding of the U2AF large subunit to pre-mRNA and to provide a bridge between the U2AF large subunit and the SR proteins.

Currently, very little is known about the detailed splicing mechanism in plants. Our recent survey revealed that most metazoan splicing factors are conserved and more than half of them are duplicated in plants (Wang and Brendel, 2004).We proposed that plants share the general splicing mechanism with metazoans but have distinct regulatory mechanisms (Wang and Brendel, 2004). Compared with nine SR proteins in humans, a total of 19 SR proteins were identified from Arabidopsis, including four families of novel SR proteins (Lazar et al., 1995; Lopato et al., 1996a, 1996b, 1999a, 1999b, 2002; Golovkin and Reddy, 1998, 1999; Kalyna and Barta, 2004; Wang and Brendel, 2004). Some novel SR proteins were found to be conserved in maize (Zea mays; Gupta et al., 2005), suggesting that these genes are possibly conserved in the plant kingdom. Recent studies using fluorescent protein tags revealed that SR proteins are dynamically distributed in nuclear speckles (Ali et al., 2003; Docquier et al., 2004; Fang et al., 2004; Lorkovic et al., 2004; Tillemans et al., 2005). The overall nuclear localization of Arabidopsis SR proteins is similar to each other and to animal homologs, although differences also exist (Tillemans et al., 2005). Overexpression of atSRp30 and atRSZ33 changes the alternative splicing pattern of some endogenous genes and causes morphological and developmental abnormalities (Lopato et al., 1999b; Kalyna et al., 2003). It is likely that atSRp30 and atRSZ33 may have important roles in ss selection. Because SR proteins function in 3'-ss recognition by binding to exonic splicing enhancers (Tian and Maniatis, 1993) and U2AF³⁵ bridges SR protein binding to exonic splicing enhancers, with U2AF⁶⁵ binding to the Py tract to stabilize the interactions (Zuo and Maniatis, 1996), it is of great interest to characterize the U2AF³⁵ homologs to understand the mechanism of 3'-ss recognition in plants.

No U2AF³⁵ homolog has been identified experimentally in plants. Database searches revealed two copies of potential U2AF³⁵ genes in Arabidopsis (Domon et al., 1998; Lorkovic and Barta, 2002; Wang and Brendel, 2004). These two genes are highly conserved with their metazoan counterparts on the sequence level, indicating that their functions may also be similar. In this study, we report the experimental characterization of the two genes. Expression pattern differences and functional divergences were found to distinguish the genes. Computational identification of U2AF³⁵ homologs in other plants revealed a highly conserved C-terminal motif specific to the plant clade of U2AF³⁵ homologs.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification of Arabidopsis U2AF Small Subunit Homologs

We recently performed a genome-scale survey of splicing-related genes in Arabidopsis and identified two U2AF³⁵ homologs (At1g27650 and At5g42820; Wang and Brendel, 2004). At1g27650 maps to the short arm of chromosome 1 and encodes a predicted polypeptide of 296 amino acids. At5g42820 maps to the long arm of chromosome 5 and encodes a predicted polypeptide of 283 amino acids. A third gene (At1g10320) shows significant similarity to human U2AF³⁵ but is presumably the ortholog of a mammalian U2AF³⁵-related sequence, U2AF1-RS1 (Kitagawa et al., 1995). The gene nomenclature conventions of previous genome-scale studies were followed and atU2AF³⁵a and atU2AF³⁵b were used to represent At1g27650 and At5g42820, respectively. At the time of this study, 16 expressed sequence tag (EST) sequences and eight cDNA sequences could be aligned to the atU2AF³⁵a region (see displays at http://www.plantgdb.org/AtGDB). Spliced alignment of the full-length cDNAs reveals a 402-nucleotide (nt) intron in the 5'-untranslated region (UTR) of atU2AF³⁵a. Two ESTs (gi2757034 and gi5839839) indicate that the 5'-UTR intron may be retained in some tissues. For atU2AF³⁵b, only three ESTs were available, and all matched to the 3'-end of the gene. One of the EST clones (accession no. AI997531) was ordered (Genome Systems) and sequenced from both directions. The full-length sequence identified the 5'-end of the atU2AF³⁵b gene, which includes a 277-nt intron in the 5'-UTR region. It also revealed a poly(A) tail at the 3'-end (which was missing in the original EST sequence). The sequence was deposited to GenBank (accession no. AF409140). As shown in Supplemental Figure 1, the atU2AF³⁵a and atU2AF³⁵b proteins contain most of the conserved domains of hsU2AF³⁵, including the RRM, one RS domain, two zinc fingers, and the two regions for interacting with the U2AF large subunit (Kielkopf et al., 2001). Both proteins lack the stretch of Glys present in hsU2AF³⁵. The sequences are overall 83% identical and each Arabidopsis homolog shows approximately 70% similarity to hsU2AF³⁵ (BLAST2 alignments using default parameters).

Expression Patterns and Alternative Splicing of atU2AF³⁵

To verify the gene structure and check the expression patterns of the atU2AF³⁵a and atU2AF³⁵b genes, specific primers were designed from the 5'- and 3'-UTRs of both genes. Reverse transcription (RT)-PCR was conducted using RNAs extracted from Arabidopsis 7-d seedlings, leaf before flowering (LeafBF), leaf after flowering (LeafAF), meristem after flowering (MeriAF), root after flowering (RootAF), stem, flower, and silique tissues. As shown in Figure 1A, both atU2AF³⁵a and atU2AF³⁵b genes express in all these tissues. No clear intron retention product can be identified for either atU2AF³⁵a or atU2AF³⁵b, indicating that the 5'-UTR intron is spliced efficiently. Intriguingly, atU2AF³⁵a seems to have noncanonical introns in addition to the 5'-UTR intron. Several smaller bands in addition to the main product were observed in the RT-PCR for atU2AF³⁵a. Sequencing results revealed that two additional segments in the 3'-end could be removed from the main transcript. These additional segments are possible introns and named alternative intron (AltIntron) 1 and AltIntron2. AltIntron1 (287 nt) and AltIntron2 (345 nt) overlap with each other. The position of these introns is shown in Figure 1B. Neither of the AltIntrons is canonical; both have a repeat region flanking the intron-exon junction (AltIntron1, AGGAGCA; AltIntron2, AAAAC). Thus their real borders are difficult to determine. Independent RT-PCRs using different transcriptase and RNA preparations confirmed the existence of additional products. Splicing of AltIntron1 and AltIntron2 removes the coding sequences for the C terminus of atU2AF³⁵a protein. The truncated Arabidopsis U2AF³⁵a proteins retain the conserved N-terminal domains and a shortened SR domain. This domain structure is similar to U2AF²⁶, a duplicated copy of U2AF³⁵ in human and mouse (Shepard et al., 2002). Whether the truncated atU2AF³⁵a proteins have physiological roles is unknown.

View larger version (63K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of atU2AF35a and atU2AF35b transcripts. A, Gel pictures of RT-PCR results. RNA tissues include seedling, LeafBF, LeafAF, MeriAF, RootAF, stem, flower, and silique. NoRT is the negative control, where equal amounts of different tissue RNAs were mixed and used in the RT-PCR reaction without adding reverse transcriptase. Genomic DNA was used as positive control. The arrows point to noncanonical alternative splicing isoforms. B, Noncanonical alternative splicing pattern. Solid boxes represent exons and lines represent introns. Numbers indicate the sizes of the corresponding introns.

To compare the expression level of atU2AF³⁵a and atU2AF³⁵b in different tissues, real-time RT-PCR was employed using primers designed from the 5'-UTR region for both genes. Because the two genes are very similar to each other at the nt level, it is not feasible to design gene-specific probes for northern analysis. To eliminate possible DNA contamination, the reverse primers were designed from the exon junctions of the 5'-UTR intron for both genes. A validation experiment using different dilutions of cDNAs confirmed that the primers for the atU2AF³⁵a and atU2AF³⁵b genes have similar amplification efficiency (data not shown). The expression of atU2AF³⁵a in LeafBF was arbitrarily selected as a calibrator and the other samples were compared with it to get relative expression levels.

As shown in Figure 2, real-time RT-PCR revealed that the atU2AF³⁵a transcript is more abundant than atU2AF³⁵b in all tissues tested. In flower, stem, silique, RootAF, and MeriAF, the atU2AF³⁵a transcript level is significantly higher than the atU2AF³⁵b level (t test, P < 0.05). atU2AF³⁵a expresses at a level over 2-fold higher than atU2AF³⁵b in these tissues. In seedlings and leaf tissues (LeafAF and LeafBF), the difference between atU2AF³⁵a and atU2AF³⁵b is not statistically significant. The atU2AF³⁵a level is less than twice that of atU2AF³⁵b in leaves. It seems that both atU2AF³⁵a and atU2AF³⁵b express at a relatively stable level in different tissues. Compared with the expression level in LeafBF, no significant difference was observed for atU2AF³⁵b in other tissues. For atU2AF³⁵a, only flower and MeriAF have significantly higher expression. From these results, we concluded that atU2AF³⁵a expresses in a level similar to or a little higher than atU2AF³⁵b in most tissues before flowering. After flowering, both genes have an increased level in leaves. The expression of atU2AF³⁵a increases significantly in meristem and flower, while expression of atU2AF³⁵b seems to be decreased in roots.

View larger version (23K): [in this window] [in a new window]   Figure 2. Expression levels of atU2AF35a and atU2AF35b. Real-time RT-PCR was performed to compare the expression levels of atU2AF35a and atU2AF35b in different tissues. The RNA tissues used are described in the legend to Figure 1. The level of atU2AF35a in leafBF was arbitrarily chosen as the calibrator and other expression levels were compared with it. Relative expression level is indicated by bar heights. The thin lines above the bars represent the SE among three experimental repeats.

Promoter::-Glucuronidase Assays Reveal Similarities and Differences between the Expression Patterns of atU2AF³⁵a and atU2AF³⁵b

RT-PCR results only revealed the expression patterns of atU2AF³⁵a and atU2AF³⁵b in major tissues. A promoter::-glucuronidase (GUS) assay was employed to identify the expression patterns in more detail. For both atU2AF³⁵a and atU2AF³⁵b, three to five independent lines transformed with two promoter constructs (the long and short ones, atU2AF³⁵a [876 and 1,358 bp]; atU2AF³⁵b [555 and 982 bp]; see "Materials and Methods" and Supplemental Fig. 2) all revealed similar GUS staining patterns, indicating that the short promoter region is a functional unit. The strong promoter control (Cauliflower mosaic virus [CaMV] 35S) showed strong GUS expression throughout the plants. No GUS activities were detected in the negative control transgenic plants (data not shown).

As shown in Figure 3, the GUS staining patterns are consistent with RT-PCR results and demonstrate that both atU2AF³⁵a and atU2AF³⁵b genes express in most tissues. The spatial and temporal expression of the two genes is similar in most tissues. Strong GUS activities are detected in 2- to 5-d seedlings (Fig. 3, A, F, and G). Shoot meristem, leaf primordia, and young leaves, including trichomes, show intense GUS activity (Fig. 3, B, C, and I–K). In large leaves, GUS expression is decreased and localized around vascular tissues of leaves (Fig. 3I). Adult leaf blades show weak GUS activity, while the vasculature and petioles have stronger GUS expression (Fig. 3, B and I). The most intense GUS activity is found in flowers. Flower buds, sepals, stamens, anthers, pollen, stigma, and the basal region of flowers all show strong GUS activity (Fig. 3, D and L–N). Petals show weak, but detectable, GUS expression. In siliques, the placenta and funiculus have strong GUS expression (Fig. 3E).

View larger version (61K): [in this window] [in a new window]   Figure 3. GUS staining patterns for atU2AF35a and atU2AF35b promoters. The tentative atU2AF35a or atU2AF35b promoters were linked to the GUS gene. A to E, M, P, and S, atU2AF35a. F to L, N, O, and T, atU2AF35b. O, R, and U, CaMV 35S promoter control. A, B, F, and G, Whole seedlings in different stages. C, I, and J, Leaves and trichomes. D, M to O, Flowers and inflorescences. E, Silique. K, Leaf primordia and meristem. L, Anther and pollen. H, P to U, Primary roots.

Both atU2AF³⁵a and atU2AF³⁵b Proteins Localize to the Cell Nucleus

As pre-mRNA splicing takes place in the nucleus, the atU2AF³⁵a and atU2AF³⁵b gene products should have nuclear localization if they are indeed splicing factors. atU2AF³⁵a and atU2AF³⁵b open reading frame (ORF) sequences were fused in frame downstream of the green fluorescent protein (GFP) coding sequence driven by the CaMV 35S promoter in gateway vector pMDC43 (Curtis and Grossniklaus, 2003). The unmodified pMDC43 was used as a control vector, with fluorescence detected in both the cell nucleus and the cytoplasm (Wang, 2005). Both atU2AF³⁵a and atU2AF³⁵b proteins are clearly enriched in nuclei and no differences were detected between them. As shown in Figure 4, strong green fluorescence was detected in the nuclei of root cells (Fig. 4C), leaf cells, guard cells (Fig. 4, D and E), and trichomes (Fig. 4F). Detailed study using confocal microscopy revealed that the distribution of atU2AF³⁵a and atU2AF³⁵b protein in the nucleus is not even (Fig. 4, A and B). They both are likely organized in nuclear speckles, a pattern similar to known SR proteins (Ali et al., 2003; Docquier et al., 2004; Lorkovic and Barta, 2004; Lorkovic et al., 2004; Tillemans et al., 2005).

View larger version (78K): [in this window] [in a new window]   Figure 4. Cellular localization of atU2AF35a and atU2AF35b proteins. The ORF of atU2AF35a or atU2AF35b was fused with GFP in frame. Nuclear localization was observed for both constructs using a fluorescence microscopy unless denoted otherwise. A and D, GFP-atU2AF35a. B and E, GFP-atU2AF35b. C and F, Both GFP-atU2AF35a and GFP-atU2AF35b. A to C, Young roots. D and E, Leaf epidermal and guard cells, with top image taken without UV excitation and bottom image taken with excitation. F, Trichome. A and B are images taken by confocal microscopy and show the uneven distribution of atU2AF35a and atU2AF35b in the nucleus. The pattern is similar to the speckle distribution of SR proteins. A GFP control showed targeting to both nucleus and cytoplasm (data not shown).

One line with T-DNA inserted into the 5'-UTR intron (SALK_050678; Alonso et al., 2003) was identified for atU2AF³⁵a. No insertion line was found for the atU2AF³⁵b gene. To knock down the atU2AF³⁵b gene, antisense and RNA interference (RNAi) vectors were constructed and transformed into Arabidopsis (vector constructions shown in Supplemental Fig. 3). Real-time RT-PCR revealed that the expression level of atU2AF³⁵a is down-regulated by 2.5-fold in atU2AF³⁵a T-DNA insertion plants (Fig. 5F). The atU2AF³⁵b RNAi plants have the atU2AF³⁵b gene knocked down to 7-fold lower expression. The atU2AF³⁵b antisense plants surprisingly have a normal atU2AF³⁵b expression level, but the atU2AF³⁵a gene is up-regulated to 2-fold. The atU2AF³⁵b gene is down-regulated relative to atU2AF³⁵a in antisense plants. Pleiotropic phenotypes were observed in the mutants and transgenic lines. The homozygous atU2AF³⁵a T-DNA insertion plants and atU2AF³⁵b transgenic plants are all late flowering under both long- and short-day conditions. As shown in Figure 5A, wild-type Arabidopsis (Col-CS60000) flowers at 20 to 25 d with 11 to 12 leaves under continuous light conditions. The atU2AF³⁵a T-DNA plants flower at 25 to 30 d with 12 to 15 leaves. The atU2AF³⁵b RNAi and antisense plants flower at 26 to 32 d with 13 to 15 leaves. In addition to the late-flowering phenotype, all three mutants have shorter flowers with enlarged bottoms compared with wild type (Fig. 5B). A few atU2AF³⁵a and atU2AF³⁵b plants show an arrested primary stem at a certain stage (Fig. 5C, indicated by a red arrow), with branches below the terminus growing normally. Distinct silique shape was observed in atU2AF³⁵a T-DNA plants. As shown in Figure 5, D and E, normal siliques are cylindrical, tapering at the ends. In contrast, the siliques in atU2AF³⁵a T-DNA plants are flattened and widened at the distal end. Additionally, silique number is increased at the terminus of the stem in atU2AF³⁵a mutants. Leaf morphology changes were also observed in atU2AF³⁵a/b plants, with atU2AF³⁵a mutants showing larger, yellowish, and flatter leaves and atU2AF³⁵b transgenic plants showing smaller, dark green, and serrated leaves (Wang, 2005).

View larger version (67K): [in this window] [in a new window]   Figure 5. Phenotypes of atU2AF35a mutant and atU2AF35b transgenic lines. Pleiotropic phenotypes are observed in atU2AF35a/b mutants. A, All atU2AF35a and atU2AF35b plants are late flowering. The gene structure and T-DNA insertion of atU2AF35a are shown above the atU2AF35a mutant. Boxes represent the exons and the thick line represents the intron. The gray rectangle denotes the protein coding region. The T-DNA insertion is indicated by a triangle. The red arrows indicate the positions of the real-time RT-PCR primers. Note that the reverse primer is flanking the exon junction. B, Flower shape changes in atU2AF35a mutants as well as in atU2AF35b plants. C, Main stem stops growing in some atU2AF35a mutants. Some atU2AF35b plants also show the same phenotype. D and E, Shape of siliques is changed in atU2AF35a mutants. F, Real-time RT-PCR results on atU2AF35a/b plants and wild type. The level of atU2AF35a in wild type was chosen as a calibrator. Bars indicate relative expression levels; thin lines above the bars represent SEs.

Because atU2AF³⁵a and atU2AF³⁵b presumably function in splicing, RT-PCR was carried out using total RNA from wild type, atU2AF³⁵a mutant, and atU2AF³⁵b transgenic lines to probe expression of 16 genes, including 12 genes predicted to be alternatively spliced, three genes (FLOWERING LOCUS C [FLC], FCA, and FPA) involved in the flowering pathway (Macknight et al., 1997; Sheldon et al., 1999; Schomburg et al., 2001), and AtDBR1 (At4g31770) as a constitutively spliced multiple-intron single-copy gene (Wang et al., 2004). No significant differences were observed for the 12 alternatively spliced genes and FPA among these plants. FLC expression levels are much higher in atU2AF³⁵b antisense plants and several minor PCR products were observed in AtDBR1 mutant and transgenic plants (Wang, 2005). The increased levels of FLC transcripts are consistent with the late-flowering phenotype in atU2AF³⁵b antisense plants. For FCA, four transcript isoforms (, , , and ) are known (see Fig. 6B), of which FCA- is the only isoform producing full-length protein (Macknight et al., 1997). The and isoforms were detected in all lines using primers designed from the UTRs, although the isoform was clearly visible only in the atU2AF³⁵b antisense (column BA in Fig. 6A) plants. As shown in Figure 6A, extra bands different from the known isoforms were produced from all mutant and transgenic lines. Three of these were cloned and sequenced, including two bands (FCA-m1 [1.4 kb] and FCA-m2 [1.2 kb]) from atU2AF³⁵b antisense and one band (FCA-m3 [547 bp]) from atU2AF³⁵b RNAi plants. Sequence alignment of the three isoforms against the genome revealed four novel introns (SI1–4) shown in Figure 6, B and C. All these introns have noncanonical intron borders, with repeated segments on both intron-exon junctions. The sequences of the repeated junctions are distinct for the different introns. Multiple exons were skipped in all novel sequenced FCA isoforms by splicing of a rather long (>4.5 kb) noncanonical intron. FCA-m1 also retained the sixteenth intron. These novel FCA isoforms demonstrate that the splicing pattern of some genes can be changed in plants with altered atU2AF³⁵a/b expression levels.

View larger version (23K): [in this window] [in a new window]   Figure 6. Novel splicing isoforms generated for the FCA gene in plants with altered levels of atU2AF35. A, RT-PCR results. M, Marker; WT, wild type; AT, atU2AF35a-TDNA plants; BR, atU2AF35b RNAi plants; BA, atU2AF35b antisense plants. Three bands indicated by the gray connectors were cloned and sequenced. B, Spliced alignment of FCA transcripts against the genome sequence. Boxes indicate exons and lines indicate introns. The four known FCA isoforms are represented in black and novel splicing isoforms are represented in gray. Vertical bars on the intron-exon junctions indicate ss different from the ones used in the FCA- isoform. Black arrows indicate the forward (F) and reverse (R) primer positions. C, Junctions of noncanonical introns. Capitalized letters indicate exon sequences and lower case letters indicate intron sequences. The unambiguous exon sequences are highlighted in gray and uncertain exon sequences are highlighted in light gray. Repeated segments are underlined.

Two U2AF³⁵ homologs are known in rice (Oryza sativa; Domon et al., 1998). We identified maize orthologs of the two rice genes by RT-PCR (Wang, 2005). All these monocot homologs have introns in the 5'-UTR, which can be alternatively spliced (Wang, 2005). By searching the EST sequences in other plant species, 15 more full-length homologs were identified, including three homologs in wheat (Triticum aestivum), two homologs in barley (Hordeum vulgare), soybean (Glycine max), tree cotton (Gossypium arboretum), and potato (Solanum tuberosum), and one homolog in Medicago (Medicago truncatula), tomato (Lycopersicon esculentum), pine (Pinus taeda), and unicellular green algae (Chlamydomonas reinhardtii). Sequences of these plant homologs are available in Supplemental File 1. As shown in Supplemental Figure 1, all these U2AF³⁵ homologs contain a degenerate RNA-binding domain, two CCCH-type zinc fingers, and one RS domain of variable length. A plant-specific conserved region was observed in the C-terminal regions. As shown in Figure 7A, U2AF³⁵ homologs from monocot plants, dicot plants, and loblolly pine contain a conserved stretch of 16 amino acids in their C terminus. The consensus sequence is SEERRA(K/R) IEQWNRERE, where underlined amino acids are completely conserved in higher plants. For the (K/R) site, all monocot homologs, except osU2AF³⁵a, have K, while most dicot homologs have R. We refer to this region as the SERE motif. Four homologs in wheat, barley, potato, and tomato (taU2AF³⁵a, hvU2AF³⁵p, stU2AF³⁵b, and leU2AF³⁵b) have two SERE motifs in their C terminus (Wang, 2005). The U2AF³⁵ homolog in unicellular green algae (crU2AF³⁵a) does not contain the SERE motif, indicating a possible function of the motif in higher plant-specific splicing mechanisms.

View larger version (35K): [in this window] [in a new window]   Figure 7. Phylogeny of U2AF35 homologs. A, C-terminal alignment of plant U2AF35 homologs. The SERE motif is indicated by a line above the alignment. Conserved amino acid residues are highlighted in black. Residues not identical, but similar to the conserved one, are highlighted in gray. The number at the end of each sequence denotes the protein length. B, Phylogenetic tree of U2AF35 homologs. Branch lengths indicate distances. Numbers on the branch are bootstrap values of confidence in the displayed branches. zm, Maize; at, Arabidopsis; os, rice; ta, wheat; hv, barley; gm, soybean; mt, Medicago truncatula; le, tomato; st, potato; ga, cotton; pt, pine; cr, unicellular algae; hs, human; dm, Drosophila melanogaster; ce, nematode; sp, fission yeast; mm, mouse; tk, bony fish; gg, chicken.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Expression and Function of U2AF³⁵ Homologs in Arabidopsis

Two homologs of U2AF³⁵ were characterized in Arabidopsis in this study. The protein sequences and domain structures of atU2AF³⁵a and atU2AF³⁵b are very similar to human U2AF³⁵, indicating that the two proteins have exchangeable functions similar to their mammalian homologs. Plants with altered levels of atU2AF³⁵a or atU2AF³⁵b showed similar phenotypes, including late flowering and flower and leaf morphology changes, suggesting that the two genes have redundant functions. Both atU2AF³⁵ genes express in all major tissues, indicating that the two homologs may function simultaneously in many cases. atU2AF³⁵a expresses at a higher level than atU2AF³⁵b in most tissues. In some tissue (e.g. root tip), however, atU2AF³⁵b expresses strongly, while atU2AF³⁵a is barely expressed, as suggested by our promoter::GUS assay. Alteration of the levels of atU2AF³⁵a and atU2AF³⁵b caused different novel splicing isoforms in FCA in the atU2AF³⁵a mutant and atU2AF³⁵b transgenic lines, while most other investigated genes were not affected. This suggests that, although most pre-mRNA splicing may not depend on the precise balance of atU2AF³⁵a and atU2AF³⁵b expression levels, some genes may require a finely tuned balance for correct splicing.

Noncanonical Alternative Splicing of U2AF³⁵ and Other Splicing Factors

RT-PCR on atU2AF³⁵a genes revealed two extra bands in addition to the constitutively spliced product. Noncanonical alternative splicing events were identified by sequencing the extra products, which in each case removes a segment with repeated borders from the second exon of the atU2AF³⁵a gene. We also found that three extra FCA isoforms were produced by excision of noncanonical introns with repeated junctions in our atU2AF³⁵ mutant and transgenic lines. Very likely the change of atU2AF³⁵ levels will lead to the usage of noncanonical sites in some pre-mRNAs. It is interesting to note that all these noncanonical introns have repeated junctions. It may be possible that some protein homodimer is involved in symmetric ss recognition when U2AF³⁵ levels are abnormal. As the expression level of atU2AF³⁵a and atU2AF³⁵b changes dynamically during growth and development, it is likely that atU2AF³⁵a can autoregulate the level of full functional protein by the noncanonical alternative splicing events. In vertebrates, the U2AF1 gene can also be alternatively spliced by inclusion of an additional exon, producing an isoform with seven amino acid differences in the RRM (Pacheco et al., 2004). Two maize SR proteins (ZmSRp31A and ZmSRp31B) also show noncanonical alternatively spliced introns (Gupta et al., 2005), strongly suggesting that many splicing factors can posttranscriptionally regulate their expression by noncanonical alternative splicing.

Evolution of U2AF³⁵ Protein Domains and U2AF³⁵ Gene Structures

Sequence alignment and phylogenetic analysis demonstrated that the U2AF³⁵ gene exists in the ancestor of eukaryotic organisms. The ancient U2AF³⁵ contained at least a RRM and two CCCH zinc fingers. It may also contain a run of Glys because the animal homologs and some monocot plant homologs have this motif. But this motif was lost in some homologs. In the plant kingdom, a C-terminal motif (SERE) was acquired after the divergence of green algae and probably before the divergence of seed plants. The SERE motif may have plant-specific functions, such as recognizing plant-specific splicing signals or interacting with plant-specific SR proteins. There might be another ancient U2AF³⁵ gene in plants, as suggested by the separation of the dicot II clade from the dicot I-monocot-ptU2AF³⁵a group, which was probably lost in monocot and some dicot (Arabidopsis) lineages. After the divergence of monocot and dicot plants, individual duplications of U2AF³⁵ genes happened in the ancestor of monocot plants, nightshade family (potato and tomato), Triticeae (wheat and barley), and Arabidopsis. The ancient plant U2AF³⁵ gene may have had introns in the 5'-UTR, as suggested by the fact that the U2AF³⁵ homologous genes in Arabidopsis, maize, and rice all have introns in the 5'-UTR. None of them have introns in either coding region or the 3'-UTR. Interestingly, the 5'-UTR intron is alternatively spliced in maize and rice homologs and the alternative splicing patterns and some intron segments are well conserved between the two monocot species (Wang, 2005), suggesting important biological functions of the 5'-UTR intron and the alternative splicing events in gene regulation.

Roles of Multiple Copies of U2AF³⁵

Plant introns have neither conserved branch sequences nor a Py tract. The 3'-ss recognition in plants will probably rely more on U2AF than in mammals. What the exact roles of plant U2AF³⁵ homologs are and how they achieve their functions are challenging questions. Similar to mammalian introns, some plant introns may be AG independent and may not require U2AF³⁵ for correct splicing. Multiple copies of U2AF³⁵ exist in nearly all higher plant genomes. Different U2AF³⁵ homologs may preferably interact with different U2AF large subunits to form different U2AF heterodimers, as suggested by variations in the interacting regions of U2AF³⁵ from the same species as well as different species (Wang, 2005). Also the RS domain and its surrounding regions of U2AF³⁵ contain many variations, indicating the flexibility of interaction between U2AF³⁵ and variable SR proteins. It is likely that some SR proteins may preferably interact with one of the U2AF³⁵ proteins, which in turn interacts with specific U2AF large subunits. The balance among different U2AF-SR protein complexes on pre-mRNAs may be important to the splicing of particular introns. Disruption of this balance will produce abnormal splicing isoforms and reduce the abundance of normal splicing isoforms, as demonstrated in FCA genes in our mutant and transgenic lines. Thus we would expect to see similar phenotypes independent of which U2AF³⁵ gene was disrupted, as observed in our mutant and transgenic lines.

Connection between Splicing and Physiological Responses in Plants

Both atU2AF³⁵a and atU2AF³⁵b mutant and transgenic lines are late flowering, with flower and leaf morphology changes. RT-PCR revealed that the FLC gene is up-regulated in atU2AF³⁵b antisense mutants (Wang, 2005). How exactly does the altered expression level of a splicing factor (atU2AF³⁵a/b) change the expression of flowering pathway genes? A possible explanation is that the production of novel FCA isoforms either reduced the level of the normal FCA isoform or possibly intervened with normal FCA isoform function, and, therefore, the FLC expression is increased. Recently, mutations disrupting the large subunit of cap-binding protein (CBP80/ABH1) were demonstrated to suppress the FRIGIDA-mediated increased FLC mRNA levels (Bezerra et al., 2004), providing another link between a splicing factor and flowering time. Knockout of the small subunit of cap-binding protein (CBP20) causes delayed development and serrated leaves (Papp et al., 2004), a phenotype very similar to our atU2AF³⁵b mutants (Wang, 2005). Both cbp20 and cbp80/abh1 mutants also confer hypersensitivity to abscisic acid. In addition, overexpression of an SR protein kinase in Arabidopsis caused delayed flowering and leaf morphology changes (Savaldi-Goldstein et al., 2003). Altered expression level of some other splicing-related genes will affect the response to drought (Xiong et al., 2001), cold (Gong et al., 2005), and salt resistance (Forment et al., 2002) in plants. It seems likely that many more connections between pre-mRNA splicing and plant responses to stress and environment are yet to be discovered.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification of U2AF³⁵ Homologs from Plants with Large Collections of EST Sequences

Plant U2AF³⁵ homologs were identified by matching Arabidopsis (Arabidopsis thaliana) homologs against plant EST sequences by BLAST. All plant ESTs were downloaded from the National Center for Biotechnology Information (NCBI) Plant Genomes Central (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/PlantList.html). Hits with E-value less than 10^–04 were regarded as significant. All hits were then used to match the EST sequences again. The same criterion was used to retrieve all related ESTs. CAP3 (Huang and Madan, 1999) was then used to cluster these related ESTs and make contigs. The contigs were matched against all known U2AF³⁵ sequences and against Arabidopsis proteins using BLASTx. Contigs were regarded as U2AF³⁵ homologs if (1) they had an E-value of less than e^–15 when matched against U2AF³⁵ homologs; and (2) their best hit in Arabidopsis is a U2AF³⁵ homolog. Putative proteins were translated from the contigs by the NCBI-ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignments of the U2AF³⁵ proteins were generated with ClustalW using default parameters (Thompson et al., 1994). The alignments were visualized using the BioEdit program (version 5.0.9; http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic analysis of the sequences was conducted using MEGA software (version 2.1; http://www.megasoftware.net; Kumar et al., 2001). The phylogenetic trees were constructed using the neighbor-joining method with a bootstrap test. The distance model used was a Kimura 2-parameter. All other parameters used were default.

Arabidopsis Growth Conditions and RNA Extraction

Arabidopsis seeds were sown in soil and grown at 4°C for 4 d, and then the plants were moved to a growth room and grown at 22°C with continuous light. Total plant RNA was isolated using either TRIzol reagent (Invitrogen) or the Plant RNeasy mini kit (Qiagen) from 0.1 to 0.2 g of different tissues. The manufacturer's protocol was followed. For Arabidopsis, root, leaf, meristem, stem, and flower tissues from wild-type ecotype Columbia were used. Total RNA was dissolved in 30 µL diethyl pyrocarbonate-treated water and saved at –20°C.

RT-PCR and Real-Time RT-PCR

The primer sequences are described in Supplemental Table I. Total RNA was treated by RQ1 RNase free DNase according to the manufacturer's protocol (Promega). Two micrograms of treated RNA were then used for first-strand synthesis and PCR according to the manufacturer's protocol (Invitrogen). A mixture of treated RNAs was used as a no-RT control. For real-time RT-PCR, PRIMER EXPRESS version 2.0 software (Applied Biosystems) was used to design oligonucleotide primers. cDNAs were prepared as described above and diluted 600-fold for amplification of the 18S ribosome RNA gene and 3-fold for other genes. One microliter of diluted cDNA was used in a 25-µL reaction with SYBR Green master mix (Applied Biosystems). All reactions were performed in triplicate by using a Prism 5700 sequence detection system (Applied Biosystems). The experiments were replicated twice using different RNA samples. Primer efficiency was checked for each primer pair by constructing a standard curve using an equal mixture of all cDNAs (Applied Biosystems). The expression level of each gene was calculated based on 2^–ct method described in user bulletin number 2 (Applied Biosystems). The relative amount of calculated message was normalized to the level of the 18S rRNA gene.

Promoter::GUS Assay

Two potential promoter regions together with the 5'-UTR region were checked for both atU2AF³⁵a and atU2AF³⁵b. For atU2AF³⁵a, promoter 1 (PGaa1) is the genomic region from 876 nt before the ATG start codon, and promoter 2 (PGaa2) is from 1,358 nt before the start codon. For atU2AF³⁵b, promoter 1 (PGab1) is from 555 nt before the start codon, and promoter 2 (PGab2) is from 982 nt before the start codon. As shown in Supplemental Figure 2, both longer promoters for atU2AF³⁵a and atU2AF³⁵b include part of the first exon of the neighboring gene. These tentative promoters were amplified from Arabidopsis genome DNA. Primers are shown in Supplemental Table I. PCR products were purified and ligated to vector pCAMBIA1381z. The vectors were subjected to sequencing from both ends to make sure the insertions were correct. In addition to the two promoters for each gene, the CaMV 35S promoter was linked with the GUS gene and used as a strong promoter control (PGxx). The empty pCAMBIA1381z (no promoter) was used as a no-promoter control (negative control, PG₀₀). These constructs are all shown in Supplemental Figure 2. The right vectors were used for Arabidopsis transformation by methods described below. Three to five individual transgenic plants from each transformation were subjected to histochemical GUS assays, following the protocol described in Weigel and Glazebrook (2002).

GFP, Antisense, and RNAi Vector Construction

GFP vectors were constructed based on vector pMDC43 (Curtis and Grossniklaus, 2003). The ORFs of atU2AF³⁵a and atU2AF³⁵b genes were amplified and cloned into the pENTR vector, then excised and transferred to the pMDC43 vector using Gateway technology (Invitrogen). Antisense vectors were constructed based on binary vector pCAMBIA1301. The vector diagrams are shown in Supplemental Figure 3. The ORF of atU2AF³⁵b was amplified by PCR, using primers described in Supplemental Table I. The PCR product was isolated, digested, and inserted into the downstream of the CaMV 35S promoter in the reverse direction. To construct an RNAi vector, the atU2AF³⁵b ORF was amplified using the primer set shown in Supplemental Table I. The PCR product and antisense vector were digested by MluI and BsrGI (New England Biolabs), then ligated by T4-DNA ligase (Promega). The RNAi construct uses the atU2AF³⁵b ORF to replace part of the antisense vector sequence in the sense direction. The resulting transcript forms a hairpin structure that triggers silencing of the endogene.

Arabidopsis Transformation

Different vectors were transformed into Agrobacterium by electroporation methods. Arabidopsis ecotype Columbia was transformed by Agrobacterium using the floral-dip method (Weigel and Glazebrook, 2002). Seeds were screened at 0.8% Arabidopsis selective medium containing 50 µg/mL hygromycin for 7 d, then transformed to 1.5% plates for another 7 d. Resistant plants were transferred to soil and analyzed.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF409139 and AF409140.

	ACKNOWLEDGMENTS

 
We would like to thank Wei Huang, Tiffanie Kuhn, and Zhen Ni Li for help with experiments. We are also grateful to Wei Huang, Dr. Philip Becraft, Dr. Robert Fluhr, and Dr. Thomas Peterson for critical reading of the manuscript. Microscopy pictures were taken in the laboratories of Dr. Shuizhang Fei and Dr. Jo Anne Powell-Coffman and in the Iowa State University Confocal Microscopy Facility.

Received November 7, 2005; returned for revision November 7, 2005; accepted November 26, 2005.

	FOOTNOTES

 
¹ This work was supported in part by the National Science Foundation (grants DBI–0110189 and DBI–0321600) and the United States-Israel Binational Agricultural Research and Development Fund (research grant no. IS–3454–03).

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: Volker Brendel (vbrendel{at}iastate.edu).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073858.

^* Corresponding author; e-mail vbrendel{at}iastate.edu; fax 515–294–6755.

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