Phylogenetic Analysis of the Plant U2 snRNP Auxiliary Factor Large Subunit A Gene Family in Response to Developmental Cues and Environmental Stimuli

Abstract

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3'-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

Keywords: U2AF65A; gene expression; protein interaction; splicing; stress.

FIGURE 1

Genomic structure organization and conserved DNA motifs amongst plant U2AF65A genes. Gene structure (A) and conserved motifs (B) of cDNA (by MEME analysis) are shown against the vertical phylogenetic tree (C).

FIGURE 2

Protein structure organization amongst plant U2AF65A genes. The protein structure (outer ring) is displayed on the outermost side, and the amino acid sequence extends from the inside to the outside, the yellow parts of the bar represent the RNA-recognition motif (RRM), and the gray parts of the bar represent the full-length amino acid sequence of the protein. A circular phylogenetic tree (inner ring) is displayed inside.

FIGURE 3

Conservative analysis of plant U2AF65A proteins. (A) Protein structures (right panel) in different plants are shown against time on the phylogenetic tree (left panel) of plants. Orange represents the RRM domain, red represents disorder, blue represents Tyr, and green represents Ser. (B) The three-dimensional structure of plant U2AF65A protein is generated (using Arabidopsis protein sequence) and represented. Blue indicates Ser and Thr in the amino acid sequence.

FIGURE 4

Analysis of the subcellular localization of OsU2AF65A. OsU2AF65A is localized to the nucleus and cytoplasm. The plasmid containing OsU2AF65A-GFP or GFP was transformed into rice protoplasts. Examination was performed under a confocal laser scanning microscope in the dark field for green fluorescence (left), white field (middle) for cell morphology and in combination (right), respectively; bar = 10 μm. Experiments were repeated three times with similar results.

FIGURE 5

Putative motifs in the promoter regions of plant U2AF65A genes. (A) Motifs are represented by rectangles of various colors. These cis-acting motifs of each plant U2AF65A gene are labeled along the 1.5-kb promoter region (straight line) according to their relative nucleotide positions to the transcript start site. (B) Specific enrichment statistics of the motifs in response to stress, hormones, and light.

FIGURE 6

Protein interacting-partners of Arabidopsis thaliana, Oryza sativa, Zea mays, Physcomitrella patens, and Chlamydomonas reinhardtii U2AF65A proteins. Protein interaction networks within species were connected by lines of corresponding colors. Red represents Physcomitrella patens, green represents Zea mays, blue represents Chlamydomonas reinhardtii, pink represents Arabidopsis thaliana, and brown represents Oryza sativa. The small orange rectangle represents the number of genes contained in the chromosome region. The outer label represents the genes encoding the interacting proteins.

FIGURE 7

Stress-inducible expression of OsU2AF65A. Three-week-old Nipponbare seedlings were exposed to 20% PEG6000, 100 mmol L^–1 sodium chloride, 100 μmol L^–1 cadmium sulfate, or cold temperature (8°C). Samples were collected at different time intervals, and the transcript level of OsU2AF65A was quantified using RT-qPCR; LOC_Os11g41820.1 was selected the U2AF65A gene in rice. Data are presented as the mean ± SD of three independent experiments and the differences amongst different time points (0, 3, 6, and 12 h) are indicated by asterisks (^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05) according to ANOVA and Tukey’s post hoc test.

FIGURE 8

AtU2AF65A mutations inhibit plant development. (A) Arabidopsis mutants were identified using RT-PCR. (B) Phenotype of Arabidopsis thaliana seedlings growing vertically for 9 days; bar = 14 mm. (C) Root length of plants (20 plants per repetition). (D) Phenotype of Arabidopsis thaliana seedlings growing horizontally for 9 days; bar = 14 mm. (E) Fresh weight of 20 Arabidopsis plants growing vertically for 9 days. (F) Dry weight of 20 Arabidopsis plants growing vertically for 9 days. Experiments in (B,D) were repeated three times with similar results. Data in (C,E,F) are presented as the means ± SD of three independent experiments, and differences between the 273/65A-1 mutants and wildtype are indicated by asterisks (^∗∗∗p < 0.001) according to ANOVA and Tukey’s post hoc test.

FIGURE 9

The U2AF65A mutations reduced the tolerance of Arabidopsis thaliana and rice plants to high temperature. (A) Hypocotyl phenotype of Arabidopsis thaliana grown vertically for 7 days at 23°C and 28°C; bar = 10 mm. (B) The hypocotyl length of plants (18 plants per replicate). (C) Growth of 3-week-old Arabidopsis seedlings exposed to high temperature for approximately 4 days; bar = 7 cm. (D) Identification of the rice U2AF65A mutant. The black column represents the exon sequence of OsU2AF65A. The 65A-11 and 65A-13 mutants lost respectively 3 bases (CGC) at position 87 and 1 base (C) at position 89 of the first exon. (E) Growth of 3-week-old rice plants before and 11 days after the high-temperature treatment; bar = 7 cm. (F) Survival rate of rice; plants were curled and withered leaves were considered dead. Experiments (A,C,E) were repeated three times with similar results. Data in (B,F) are presented as the means ± SD of three independent experiments, and differences between the mutants and wildtype are indicated by asterisks (^∗∗∗p < 0.001) according to ANOVA and Tukey’s post hoc test.