The Rice Serine/Arginine Splicing Factor RS33 Regulates Pre-mRNA Splicing during Abiotic Stress Responses

Abstract

Abiotic stresses profoundly affect plant growth and development and limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich splicing factors play a key role in pre-mRNA splicing to regulate different biological processes under stress conditions. Alternative splicing (AS) of SR transcripts and other transcripts of stress-responsive genes generates multiple splice isoforms that contribute to protein diversity, modulate gene expression, and affect plant stress tolerance. Here, we investigated the function of the plant-specific SR protein RS33 in regulating pre-mRNA splicing and abiotic stress responses in rice. The loss-of-function mutant rs33 showed increased sensitivity to salt and low-temperature stresses. Genome-wide analyses of gene expression and splicing in wild-type and rs33 seedlings subjected to these stresses identified multiple splice isoforms of stress-responsive genes whose AS are regulated by RS33. The number of RS33-regulated genes was much higher under low-temperature stress than under salt stress. Our results suggest that the plant-specific splicing factor RS33 plays a crucial role during plant responses to abiotic stresses.

Keywords: SR proteins; abiotic stress; alternative splicing; genome engineering; pre-mRNA splicing.

Figure 1

Genome-wide effects of the loss of OsRS33 on alternative splicing in rice. (a) Targeted mutagenesis of the OsRS33 (LOC_Os02g03040) locus in rice [24]. RS33 is a plant-specific SR protein characterized by the RNA Recognition Motif (RRM) and arginine–serine (RS) repeat domains. The rs33 knockout mutant has an 11-nt deletion and a 1-nt substitution. (b) The number of up- and downregulated differentially expressed genes (DEGs) between the WT and rs33. (c) Bar plot showing the number of each type of differential alternative splicing (DAS) event mediated by RS33; the majority of these AS events were intron retention (IR) events. SE, exon skipping; A5′S, 5′ alternative splice site; A3′S, 3′ alternative splice site. (d) Venn diagram displaying the overlap between DEGs and DAS genes. (e) cDNA was prepared from one-week-old WT and rs33 seedlings. RT-PCR was performed using primers that flank introns subject to AS in selected genes. Intron retention was observed in rs33 mutant rice plants. Arrowheads indicate splicing variants. The gene structures and retained introns are shown. Red boxes indicate the PCR fragments.

Figure 2

The rs33 mutant is hypersensitive to salt stress. (a,b) Seeds of rs33 and WT were germinated for ten days in ½ MS media supplemented with 0 mM and 100 mM NaCl. Germination was severely inhibited in rs33 compared to WT seeds. Other growth parameters—shoot length (c) and seedling fresh weight (d)—were also severely affected in rs33 as compared to WT seedlings. In c, d; significance is represented by letters. All the variables with the same letters are not statistically significant. If two variables have different letters, they are significantly different.

Figure 3

The rs33 mutant is hypersensitive to cold stress. (a) The rs33 mutant and WT seeds were germinated for ten days at the control temperature (27 °C) or different low temperatures (20 °C, 14 °C, and 4 °C). For 14 °C, the seeds were first kept at 14 °C for seven days and then transferred to 27 °C for 4 days. For 4 °C, the seeds were initially treated at 4 °C for four days and then germinated at 27 °C for seven days. At 20 °C, the germination of the rs33 seeds was significantly delayed compared to that of the WT. The shoot length (b) and seedling fresh weight (c) were significantly affected in rs33 as compared to WT seedlings. In b, c; all the variables with the same letters are not statistically significant. If two variables have different letters, they are significantly different.

Figure 4

Genome-wide effects of the loss of OsRS33 on gene expression and RNA splicing under salt stress. (a) Number of differentially expressed genes (DEGs) between the rs33 mutant and the WT. One-week-old rice seedlings were treated with 200 mM NaCl for 3 and 6 h and used for RNA extraction. A high number of DEGs was observed for rs33 seedlings after a 6-h treatment with NaCl. (b) Clustering analysis of the DEGs. Cluster 1 clearly shows the downregulation of translation/ribosome biogenesis genes in rs33 in the 6-h NaCl treatment. Cluster 3 shows the upregulation of stress-responsive genes in rs33 in the 6-h salt treatment as compared to the WT. (c) Bar plot showing the number of each type of differential alternative splicing (DAS) event induced by salt treatment in the rs33 mutant and the WT. (d) Heatmap and hierarchical clustering of DAS genes after salt treatment in rs33 and WT. (e) Venn diagram displaying the overlap between DEGs and DAS genes. (f) cDNA was prepared from one-week-old WT and rs33 seedlings treated with 200 mM NaCl for 3 and 6 h. Mock-treated samples were used as controls. RT-PCR was performed using primers that flank introns subject to AS in the selected genes. Arrowheads indicate splicing variants. The gene structures and retained introns are shown. Red boxes indicate the PCR fragments. C3; control 3 h, C6; control 6 h, S3; salt 3 h, and S6; salt 6 h.

Figure 5

Genome-wide effects of the loss of OsRS33 on gene expression and RNA splicing under low-temperature stress. (a) A number of differentially expressed genes (DEGs) between the rs33 mutant and the WT. One-week-old rice seedlings were treated with low-temperature stress (4 °C) for 3 and 6 h and used for RNA extraction. (b) Clustering analysis of the DEGs. Cluster 1 shows the upregulation of oxidative stress response genes in rs33 after 6 h of cold treatment as compared to the WT. Cluster 4 shows that the cell wall biogenesis genes were downregulated after 6 h of cold treatment in rs33. (c) Bar plot showing the number of each type of differential alternative splicing (DAS) event induced by low-temperature stress in the rs33 mutant and the WT. (d) Heatmap and hierarchical clustering of DAS genes after the cold treatment in rs33 and WT. Cluster 1 contains stress-related genes with quick AS regulation upon stress. Cluster 5 indicates that some genes of the lipid metabolism pathways do not respond to cold in rs33. (e) Venn diagram displaying the overlap between DEGs and DAS genes. (f) cDNA was prepared from one-week-old WT and rs33 seedlings treated at 4 °C for 3 and 6 h. Mock-treated samples were used as the controls. RT-PCR was performed using primers that flank introns subject to AS in the selected genes. Arrowheads indicate splicing variants. The gene structures and retained introns are shown. Red boxes indicate the PCR fragments. C3; control 3 h, C6; control 6 h, F3; 4 °C 3 h, and F6; 4 °C 6 h.