The splicing auxiliary factor OsU2AF35a enhances thermotolerance via protein separation and promoting proper splicing of OsHSA32 pre-mRNA in rice

Abstract

Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (U2 snRNP auxiliary factor) is an essential splicing complex with critical roles in recognizing the 3'-splice site of precursor messenger RNAs (pre-mRNAs). The U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 snRNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the OsU2AF35a gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid-liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA-seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in osu2af35a-1 were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre-mRNAs of a considerable number of genes in the osu2af35a-1 mutant exhibited defective splicing, among which was the OsHSA32 gene. Knocking out OsHSA32 significantly reduced the thermotolerance of rice, while overexpressing OsHSA32 could partially rescue the heat sensitivity of osu2af35a-1. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre-mRNA splicing.

Keywords: OsHSA32; OsU2AF35a; condensation; liquid–liquid phase separation; pre‐mRNA splicing; rice thermotolerance.

Figure 1

Expression patterns of OsU2AF35a. (a–d) RT‐qPCR analysis of OsU2AFs' expression levels at different time after heat stress (45 °C). The wild‐type ZH11 seedlings at 2.5‐ to 3.5‐leaf stage were used for treatment and the roots were harvested for gene expression detection. (e) Up‐regulation of OsU2AF35a by ABA and abiotic stresses. The wild‐type ZH11 seedlings at 2.5‐ to 3.5‐leaf stage were treated with various stimuli (100 mM ABA; NaCl, 120 mM; PEG6000, 20%; Heat, 45 °C; Cold, 4 °C) and the seedlings were harvested for gene expression detection. (f) Expression analysis of OsU2AF35a in different tissues containing root, shoot, stem, leaf sheath, leaf, young panicle, mature panicle and flag leaf by RT‐qPCR. OsActin1 was used as an internal control. Data represent means ± SE; n = 3. Different letters above the bars indicate differences determined by one‐way ANOVA with Tukey's HSD post‐hoc test at P < 0.05.

Figure 2

OsU2AF35a positively regulates rice thermotolerance at seedling and reproductive stages. (a) Heat stress phenotypes of the osu2af35a mutants. The seedlings (3.5‐ to 4.5‐leaf stage) of ZH11 and two osu2af35a mutants grown at 28 °C were transferred to 45 °C for 24 h and then photographed after recovering at 28 °C for 7 days. Scale bars = 12.5 cm. (b) Statistical analysis of survival rate in (a) after heat treatment and recovery based on the appearance of newly developed green leaves. (c) Electrolytic leakage in ZH11 and osu2af35a mutant plants before and after heat treatment. Seedlings at 2.5‐ to 3.5‐leaf stage were transferred to 45 °C for 6 h, leaves were harvested for electrolyte measurement. (d) Heat stress phenotypes of the OsU2AF35a‐OE lines. The seedlings (3.5‐ to 4.5‐leaf stage) of ZH11 and two OsU2AF35a‐OE lines grown at 28 °C were transferred to 45 °C for 48 h and then photographed after recovering at 28 °C for 7 days. Scale bars = 12.5 cm. (e) Statistical analysis of survival rate in (d) after heat treatment and recovery based on the appearance of newly developed green leaves. (f) Electrolytic leakage in ZH11 and OsU2AF35a‐OE plants before and after heat treatment. Seedlings at 2.5‐ to 3.5‐leaf stage were transferred to 45 °C for 6 h, leaves were harvested for electrolyte measurements. (g–j) Comparison of thermotolerance among ZH11, osu2af35a mutant, and OsU2AF35a‐OE plants at the reproductive stage in the phytotron. The above‐mentioned plants grown at 28 °C were subjected to heat stress (40 °C in the light for 12 h and 31 °C in the dark for 12 h) at flowering stage for 7 days, and then recovered under normal conditions until seed maturation and panicles were photographed (g), seed setting rate (h), 1000‐grain weight (i), and grain yield per plant (j) were measured. The control plants were constantly placed at normal growth temperature. In (b) and (e), data represent means ± SE, n = 4 (****P < 0.0001, Student's t‐test). In (c), (f), and (h–j), data represent means ± SE, n = 3 or 12. Different letters above the bars indicate differences determined by one‐way ANOVA with Tukey's HSD post‐hoc test at P < 0.05.

Figure 3

OsU2AF35a interacts with the RRM3 domain of OsU2AF65a. (a) Schematic diagram of protein structure and IDR prediction of OsU2AF35a/65a. (b) Interaction between OsU2AF35a and OsU2AF65a was analysed with Y2H system. Transformants were photographed after 5 days of growth on medium lacking Trp and Leu, or lacking Trp, Leu, His, and Ade. (c) BiFC assay for the interaction of OsU2AF35a and OsU2AF65a. Fluorescence was observed in the nuclear compartment of transformed tobacco (N. benthamiana) cells, resulting from the complementation of OsU2AF35a‐nYFP+OsU2AF65a‐cYFP or OsU2AF35a‐cYFP+OsU2AF65a‐nYFP. No signal was obtained for the negative controls in which OsU2AF35a‐nYFP was co‐expressed with cYFP, or nYFP was co‐expressed OsU2AF65a‐cYFP. YFP signal was detected by confocal microscopy. (d) Interaction test between OsU2AF35a and different truncated OsU2AF65a with specific deletion in yeast. Transformants were photographed after 5 days of growth on medium lacking Trp and Leu, or lacking Trp, Leu, His, and Ade.

Figure 4

OsU2AF35a undergoes reversible and dynamic condensation in response to heat stress. (a) Subcellular localization of OsU2AF35a‐EGFP, N187aa‐EGFP, and IDR‐EGFP in tobacco leaves. U170K‐mCherry, a spliceosome component marker localized in nucleus. (b) Representative confocal microscopic images of tobacco epidermal cells expressing OsU2AF35a or OsU2AF35a truncations. The tobacco plants were treated with or without 37 °C for 15 min. (c) Representative confocal microscopic images of tobacco epidermal cells expressing OsU2AF35a‐EGFP. The tobacco plants were treated as indicated. (d–e) Representative confocal microscopic images of rice root tip cells expressing OsU2AF35a‐EGFP or N187aa‐EGFP. The transgenic rice seedlings were treated as indicated. (f) FRAP of OsU2AF35a‐EGFP nuclear condensates formed in rice root tips. Time 0 s indicates the time of the photobleaching pulse. (g) Plot showing the time course of the recovery after photobleaching OsU2AF35a nuclear condensates. Data represent means ± SE; n = 3.

Figure 5

OsU2AF35a undergoes LLPS in vitro. (a) LLPS of purified EGFP‐OsU2AF35a‐6 × His protein under micromolar protein concentrations. LLPS condition: 120 mM NaCl, pH 7.5 and 15% PEG 8000. (b) Representative confocal microscopic images showing droplet formation of 20 μM EGFP‐OsU2AF35a‐6 × His protein as temperature increases from 28 °C to 45 °C under 120 mM NaCl and pH7.5. Addition of 1 volume of 5% 1,6‐hexanediol solution disrupts OsU2AF35a phase separation, but not the mock (water). (c, d) Fusion (c) and fission (d) of two EGFP‐OsU2AF35a‐6 × His droplets. Time points are indicated in seconds. (e) FRAP of EGFP‐OsU2AF35a‐6 × His droplets. Time 0 s indicates the time of the photobleaching pulse. (f) Plot showing the time course of the recovery after photobleaching EGFP‐OsU2AF35a‐6 × His droplets. Data are presented as the mean ± SE; n = 3.

Figure 6

OsU2AF35a condensation indispensable for heat stress tolerance. (a) The heat stress phenotypes of indicated genotypes. The seedlings (3.5‐ to 4.5‐leaf stage) of all genotypes grown at 28 °C were transferred to 45 °C for 24 h and then photographed after recovering at 28 °C for 7 days. Scale bars = 12.5 cm. (b) Statistical analysis of survival rate in (A) after heat treatment and recovery based on the appearance of newly developed green leaves. (c) Electrolytic leakage of indicated genotypes before and after heat treatment. Seedlings at 2.5‐ to 3.5‐leaf stage were transferred to 45 °C for 6 h, leaves were harvested for electrolyte measurements. In (b, c), data represent means ± SE, n = 3. Different letters above the bars indicate differences determined by one‐way ANOVA with Tukey's HSD post‐hoc test at P < 0.05.

Figure 7

The osu2af35a‐1 mutation causes disrupted gene expression as determined in RNA‐seq experiments. (a) Summary of differentially expressed genes in osu2af35a‐1. Criteria for differential expression were set as q ≤ 0.05, fold change (FC) ≥ 2 for upregulation or FC ≤0.5 for down‐regulation. (b) Heat map of differentially expressed genes in osu2af35a‐1. Samples (rows) and genes (column) are hierarchically clustered via Pearson correlation. (c) GO term distribution of differentially expressed genes in osu2af35a‐1. (d) Validation of differential gene expression portion of the RNA‐seq results by RT‐qPCR analysis. The ZH11 and osu2af35a‐1 mutant plants at 2.5‐ to 3.5‐leaf stage were treated at 45 °C for 9 h, and total RNA was extracted for RT‐qPCR. OsActin1 was used as an internal control. Data represent means ± SE; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant at P < 0.05; Student's t‐test).

Figure 8

The osu2af35a‐1 mutation causes defects in alternative splicing of gene transcripts. (a) Summary of genes whose transcripts were abnormally spliced in osu2af35a‐1 as determined by RNA‐seq experiments. (b) Genes with defects in different types of alternative splicing patterns in osu2af35a‐1 as determined by RNA‐seq experiments. (c) GO term distribution of abnormally spliced genes in osu2af35a‐1. (d) Validation of intron‐retention and exon‐skipping events in ZH11, osu2af35a‐1, and osu2af35a‐2 plants as determined by RT‐qPCR analysis. Red font indicates the bands that appear after mis‐splicing. (e) The retention of intron 1 causes the premature occurrence of the stop codon in OsHSA32. The green sequences represent exon 1 and exon 2, the black sequence indicates intron 1, and the red sequence denotes the stop codon. (f) Validation of intron 1 retention of OsHSA32 transcripts in the indicated lines through RT‐PCR analysis. Red font represents the bands that appear after mis‐splicing.

Figure 9

Overexpressing OsHSA32 partially rescues the heat sensitivity of osu2af35a mutant. (a) Heat stress phenotypes of the oshsa32 mutants. The seedlings (3.5‐ to 4.5‐leaf stage) of ZH11 and two oshsa32 mutants grown at 28 °C were transferred to 45 °C for 24 h and then photographed after recovering at 28 °C for 7 days. Scale bars = 12.5 cm. (b) Statistical analysis of survival rate in (a) after heat treatment and recovery based on the appearance of newly developed green leaves. Data represent means ± SE; n = 3 (****P < 0.0001; Student's t‐test). (c) Electrolytic leakage in ZH11 and oshsa32 mutant plants before and after heat treatment. Seedlings at 2.5‐ to 3.5‐leaf stage were transferred to 45 °C for 9 h, leaves were harvested for electrolyte measurements. (d) RT‐qPCR analysis of OsHSA32 expression in osu2af35a‐1 and two OsHSA32/osu2af35a‐1 (#7 and #8) lines. OsActin1 gene was used as an internal control. Data represent means ± SE; n = 3. (e) Heat stress phenotypes of the OsHSA32/osu2af35a‐1 lines. The seedlings (3.5‐ to 4.5‐leaf stage) of ZH11, osu2af35a‐1, and two OsHSA32/osu2af35a‐1 lines grown at 28 °C were transferred to 45 °C for 24 h and then photographed after recovering at 28 °C for 7 days. Scale bars = 12.5 cm. (f) Statistical analysis of survival rate in (e) after heat treatment and recovery based on the appearance of newly developed green leaves. In (c, d) and (f), data represent means ± SE, n = 3. Different letters above the bars indicate differences determined by one‐way ANOVA with Tukey's HSD post‐hoc test at P < 0.05.

Figure 10

A potential working model for OsU2AF35a during heat response. Under normal growth condition, OsU2AF35a is diffused in the nucleus. When rice is subjected to high temperature stress, OsU2AF35a protein forms aggregates through LLPS. Post‐phase separation, OsU2AF35a can more effectively promote the accurate splicing of the pre‐mRNA of OsHSA32 gene, ultimately improving the thermotolerance of rice.