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Plant Physiol. 2006 Feb; 140(2): 624–636.
PMCID: PMC1361329

Molecular Characterization and Phylogeny of U2AF35 Homologs in Plants1,[W][OA]


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::β-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.

Splicing is an essential process in eukaryotic gene expression. The precise excision of introns from pre-mRNA requires a dynamically assembled RNA protein complex (spliceosome). Many proteins participate in intron and exon definition prior to the assembly of U1 and U2 small nuclear RNP (snRNP). U2AF is such a splicing factor. Before spliceosome assembly, U2AF binds to the polypyrimidine (Py) tract between the intron branch-point and the 3′-AG dinucleotide intron boundary to recruit U2 snRNP to the branch-point sequence. The U2AF protein is composed of a large subunit (U2AF65) and a small subunit (U2AF35; Zamore and Green, 1989), with U2AF65 binding directly to the Py tract (Zamore et al., 1992) and U2AF35 binding to the 3′-AG boundary (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999). U2AF35 can promote binding of U2AF65 to the Py tract by interacting simultaneously with the U2AF65 and arginine-serine (SR) proteins (Zuo and Maniatis, 1996). It was also shown that the binding of U2AF35 can trigger events in spliceosome assembly in addition to stabilizing U2AF65 binding (Guth et al., 2001). In vivo studies in Drosophila revealed that U2AF38 is an essential factor because mutations in U2AF38 caused lethality and development defects (Rudner et al., 1996). In vitro studies, however, suggested that some pre-mRNAs, including human β-globin pre-mRNA and adenovirus major late pre-mRNA, do not require U2AF35 in splicing (Guth et al., 1999). These seemingly conflicting results indicate that U2AF35 may function in a substrate-specific manner.

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 (ψRRM) flanked by two highly conserved C8C5C3H zinc fingers (Kielkopf et al., 2001, 2004). The ψRRM 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 ψRRM, 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.


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 ψRRM, 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.

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 ...

atU2AF35a Has Higher Expression Levels Than atU2AF35b in Most Tissues

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.

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 ...

Promoter::β-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::β-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).

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 ...

Differences between the expression of atU2AF35a and atU2AF35b were also discovered in flowers and young roots. As shown in Figure 3, M to Q, strong GUS activities were detected on the tops of pistils in control plants (CaMV 35S). The atU2AF35a transformants also have detectable GUS expression. atU2AF35b transformants, however, have no clear GUS activities. A distinct expression pattern was discovered in young roots. For atU2AF35a, expression in young roots is limited to vascular regions (Fig. 3P). Root tips and hairs do not show clear GUS expression (Fig. 3S). The atU2AF35b and CaMV 35S promoter drive strong GUS activities on the whole root (Fig. 3, Q and R), with the strongest expression in root tips (Fig. 3, T and U).

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).

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-atU2AF ...

Plants with Altered Expression Levels of atU2AF35 Genes Show Pleiotropic Phenotypes

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).

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 ...

Noncanonical Splicing Patterns in Transgenic Plants

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 (α, β, γ, and δ) are known (see Fig. 6B), of which FCA-γ is the only isoform producing full-length protein (Macknight et al., 1997). The γ and δ isoforms were detected in all lines using primers designed from the UTRs, although the δ 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.

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 ...

Motifs and Molecular Phylogeny of Plant U2AF35 Homologs

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.

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, ...

A phylogenetic tree was constructed based on the multiple sequence alignment of the N-terminal regions upstream of the RS domain shown in Supplemental Figure 1. As shown in Figure 7B, all plant homologs cluster into one group and all animal homologs cluster into another group. In the plant group, the seed plant homologs separate into four clades, including two monocot and two dicot clades. The green algae homolog appears as an outgroup. zmU2AF35b (maize), osU2AF35b (rice), hvU2AF35a (barley), taU2AF35b and taU2AF35c (wheat) group into the monocot I clade, and zmU2AF35a, osU2AF35a, hvU2AF35p, and taU2AF35a group into the monocot II clade. For dicot plants, atU2AF35a and atU2AF35b (Arabidopsis), gmU2AF35b (soybean), and gaU2AF35b (tree cotton) group into the dicot I clade, and the remaining dicot homologs cluster into the dicot II clade. The dicot I clade is clustered in a big group with the monocot clades and ptU2AF35a (from lobolly pine), indicating the ancient form of U2AF35 in the ancestor of seed plants. It seems that the ancient U2AF35 gene likely had introns in the 5′-UTR, as suggested by the gene structure of known homologs in Arabidopsis, rice, and maize. atU2AF35a and atU2AF35b both fall into the dicot I clade, indicating a recent duplication event in Arabidopsis.


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 ψRRM (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 ψRRM 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.


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−ΔΔct 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.

Supplementary Material

Supplemental Data:


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.


1This 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 (ude.etatsai@lednerbv).

[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.


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