Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

doi:10.1016/j.rsci.2020.05.005

Abstract

Abstract:

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences, and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations. In this study, we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene. Cloning of OsRAD1 cDNAs revealed three transcript variants, named as OsRAD1.1, OsRAD1.2 and OsRAD1.3, respectively, which were derived from alternative splicing of the last intron. Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain. The three transcripts exhibited similar expression patterns in various tissues, but the expression level of OsRAD1.1 was the highest. Specific knockout of OsRAD1.1 led to sterility, while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility. Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility. Yeast two-hybrid assay showed that OsRAD1.1, but not OsRAD1.2 and OsRAD1.3, interacted with the three other meiosis proteins OsHUS1, OsRAD9 and OsRAD17, suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.

Key words: Oryza sativa, OsRAD1, alternative splicing, meiosis, protein interaction, fertility, sterility

Shuting Yuan, Chunjue Xu, Wei Yan, Zhenyi Chang, Xingwang Deng, Zhufeng Chen, Jianxin Wu, Xiaoyan Tang. Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis[J]. Rice Science, 2020, 27(4): 289-301.

Figures/Tables 15

Supplemental Table 1. Primers used in this study.

Primer	Forward (5'-3')	Reverse('5'-3')
OsRAD1-P1	CTCAACAGCCAAGCCGAA	AGGTGTGGAACGGTGGAA
OsRAD1-P2	AGAGGGCGCGAAACCGTAGA	TCATACCACAATCCCTACGCATCAT
OsRAD1-P3	AGAGGGCGCGAAACCGTAGA	AAAAACGGAGTCAAGCAATCATCAT
OsRAD1-P4	TCCCCATCCTCTCCGTCGACTTCGT	ACTCAAGTACTCTGTGGAGGATGCCA
CR-1 target	CAAGGTCTACCTCAAGCGTG
CR-2 target	CAGAACTTCCGTAATGTTGC
CR-3 target	ACTGTTGCTTGAGCAGCAAA
OsRAD1-P5	GATCGAGGACCTTGAGTGGC	CACCTTGCTCCCACGATTCT
OsRAD1-P6	GATGTTGGCGACCTCGTATT	CGCACAATCCCACTATCCTT
OsRAD1-P7	GCCCAAATTGAATTGAAATCTGGCCTTTGAAGA	GCCCAAATTGAATTGAAATCTGGCCTTTGAAGA
OsRAD1-P8	CATGAACAGATCTGCCCCGA	AGCCACAAAACCCCATCGAA
OsRAD1-P9	CAGAACTTCCGTAATGTTGCTGG	TCGGTTTTGCCACTACCAGT
OsRAD1-P10	GGGCCCAAAGCATTTTCCTT	TCCAAAAACGGAGTCAAGCA
OsRAD1-P11	TGGTAGGGGTGGGATGCTTA	ACCACAATCCCTACGCATCA
SP-LR	GCGCGGTGTCATCTATGTTACT	CCCGACATAGATGCAATAACTTC
OsACTIN1-qPCR	GCTATGTACGTCGCCATCCA	GGACAGTGTGGCTGACACCAT
OsRAD1-qPCR	GATCGAGGACCTTGAGTGGC	AGGGGAACTCAATCTGCAAGT
OsRAD1.1-qPCR	AGAATCGTGGGAGCAAGGTG	AGCCCCTCCAGCAACATTAC
OsRAD1.2+3-qPCR	AGAATCGTGGGAGCAAGGTG	GGAAAATGCTTTGGGCCCTC
OsRAD1.2-qPCR	GTTGCAAGGCCAGGGTGTAA	GGTTCACCACGCTGTAGACT
OsRAD1.3-qPCR	CAAGGCCAGGTGTTCAAACG	GGTTCACCACGCTGTAGACT
MEL1-qPCR	TTACCCCCATTCCTATGAGCC	CAATCTTGCACACCTCCATAG
ZEP1-qPCR	CTGCCTCCAACATTAGTCAGC	CACTCGACCTAGAAGCTCCTG
PAIR1-qPCR	GGATGGACCCAGATTAACC	CTGTTTAGGTGCCACCCTGT
PAIR2-qPCR	TGCCAGAGGAGAGGACCATTC	CACGAGATGCTTGCTATTGAC
PAIR3-qPCR	GGAAGTTGAGCTGACGAACA	CAGTTCCCTGAGACAAGTTC
OsRAD1.1-BD	ATGAGCTCGTCGACGTCC	CTACGCATCATTTATCTCATA
OsRAD1.2-BD	ATGAGCTCGTCGACGTCC	TCACCACGCTGTAGACTGTT
OsRAD1.3-BD	ATGAGCTCGTCGACGTCC	TCACCACGCTGTAGACTGTT
OsRAD1M-BD	ATGAGCTCGTCGACGTCC	CTAAGCAATTCGGCTTGGCT
OsHUS1-AD	ATGAAGTTCAAGGCCTTCTT	TTAACTGCCAGGGTCAAGGA
OsRAD9-AD	ATGGAGCTGTCTATGAGCGG	CTAGTCCATGTAGTGCGGTG
OsRAD17-AD	ATGGGGAAGCGGCCGCCGGT	TCACCAATCTTCTATCTCAT

Supplemental Table 1. Primers used in this study.

Primer	Forward (5'-3')	Reverse('5'-3')
OsRAD1-P1	CTCAACAGCCAAGCCGAA	AGGTGTGGAACGGTGGAA
OsRAD1-P2	AGAGGGCGCGAAACCGTAGA	TCATACCACAATCCCTACGCATCAT
OsRAD1-P3	AGAGGGCGCGAAACCGTAGA	AAAAACGGAGTCAAGCAATCATCAT
OsRAD1-P4	TCCCCATCCTCTCCGTCGACTTCGT	ACTCAAGTACTCTGTGGAGGATGCCA
CR-1 target	CAAGGTCTACCTCAAGCGTG
CR-2 target	CAGAACTTCCGTAATGTTGC
CR-3 target	ACTGTTGCTTGAGCAGCAAA
OsRAD1-P5	GATCGAGGACCTTGAGTGGC	CACCTTGCTCCCACGATTCT
OsRAD1-P6	GATGTTGGCGACCTCGTATT	CGCACAATCCCACTATCCTT
OsRAD1-P7	GCCCAAATTGAATTGAAATCTGGCCTTTGAAGA	GCCCAAATTGAATTGAAATCTGGCCTTTGAAGA
OsRAD1-P8	CATGAACAGATCTGCCCCGA	AGCCACAAAACCCCATCGAA
OsRAD1-P9	CAGAACTTCCGTAATGTTGCTGG	TCGGTTTTGCCACTACCAGT
OsRAD1-P10	GGGCCCAAAGCATTTTCCTT	TCCAAAAACGGAGTCAAGCA
OsRAD1-P11	TGGTAGGGGTGGGATGCTTA	ACCACAATCCCTACGCATCA
SP-LR	GCGCGGTGTCATCTATGTTACT	CCCGACATAGATGCAATAACTTC
OsACTIN1-qPCR	GCTATGTACGTCGCCATCCA	GGACAGTGTGGCTGACACCAT
OsRAD1-qPCR	GATCGAGGACCTTGAGTGGC	AGGGGAACTCAATCTGCAAGT
OsRAD1.1-qPCR	AGAATCGTGGGAGCAAGGTG	AGCCCCTCCAGCAACATTAC
OsRAD1.2+3-qPCR	AGAATCGTGGGAGCAAGGTG	GGAAAATGCTTTGGGCCCTC
OsRAD1.2-qPCR	GTTGCAAGGCCAGGGTGTAA	GGTTCACCACGCTGTAGACT
OsRAD1.3-qPCR	CAAGGCCAGGTGTTCAAACG	GGTTCACCACGCTGTAGACT
MEL1-qPCR	TTACCCCCATTCCTATGAGCC	CAATCTTGCACACCTCCATAG
ZEP1-qPCR	CTGCCTCCAACATTAGTCAGC	CACTCGACCTAGAAGCTCCTG
PAIR1-qPCR	GGATGGACCCAGATTAACC	CTGTTTAGGTGCCACCCTGT
PAIR2-qPCR	TGCCAGAGGAGAGGACCATTC	CACGAGATGCTTGCTATTGAC
PAIR3-qPCR	GGAAGTTGAGCTGACGAACA	CAGTTCCCTGAGACAAGTTC
OsRAD1.1-BD	ATGAGCTCGTCGACGTCC	CTACGCATCATTTATCTCATA
OsRAD1.2-BD	ATGAGCTCGTCGACGTCC	TCACCACGCTGTAGACTGTT
OsRAD1.3-BD	ATGAGCTCGTCGACGTCC	TCACCACGCTGTAGACTGTT
OsRAD1M-BD	ATGAGCTCGTCGACGTCC	CTAAGCAATTCGGCTTGGCT
OsHUS1-AD	ATGAAGTTCAAGGCCTTCTT	TTAACTGCCAGGGTCAAGGA
OsRAD9-AD	ATGGAGCTGTCTATGAGCGG	CTAGTCCATGTAGTGCGGTG
OsRAD17-AD	ATGGGGAAGCGGCCGCCGGT	TCACCAATCTTCTATCTCAT

Fig. 1. Phenotypes of Huanghuazhan wild type (WT) and m10690 mutant plants.A, WT and m10690 mutant plants after heading. Scale bar, 10 cm. B, Seed-setting in WT and the mutant. Scale bar, 3 cm. C, Seed-setting of osnp1 and m10690 plants at 20 d after pollinated with the WT pollen. Red arrow indicates the seed-setting in the mutant. Scale bar, 2 cm. D, WT and m10690 spikelets with the palea and lemma removed to show the anthers. Scale bar, 1 mm. E, WT pollen grains stained with I2-KI. Scale bar, 100 μm. F, m10690 pollen grains stained with I2-KI. Scale bar, 100 μm.

Fig. 2. Transverse sections of anther in Huanghuazhan wild type (WT) and m10690 plants.WT anther sections are shown in A, C, E, G, I, K and M. The m10690 mutant anther sections are shown in B, D, F, H, J, L and N.E, Epidermis; En, Endothecium; M, Middle layer; T, Tapetal layer; PMC, Pollen mother cell; Dys, Dyads; Tds, Tetrads; MP, Mature pollen; Msp, Microspores. Scale bars, 20 μm.

Supplemental Fig. 1. Scanning electron microscopy analysis of pollen grains, inner surface of tapetum, and outer surface of anther wall in the WT and m10690 mutant.Pollen grain of wild-type (WT) (A) and m10690 mutant (B), inner surface of WT tapetum (C) and m10690 mutant tapetum (D), and out surface of WT anther wall (E) and m10690 mutant anther wall (F) are shown. Scale=10μm.

Fig. 3. Chromosome behavior during meiosis in pollen mother cells of Huanghuazhan wild type (WT) and m10690.A?F, Different meiosis stages for the WT. G?L, Different meiosis stages for the m10690 mutant. Red arrows indicate broken chromosome fragments. Scale bars, 10 μm.

Fig. 4. Development of embryo sac in Huanghuazhan wild type (WT) and m10690 plants.A?J, The WT embryo sacs. K?O, The m10690 mutant embryo sacs. Arrows indicate the megaspores at various developmental stages. Scale bars, 20 μm.

Supplemental Fig. 2. Expression of meiosis genes acting at prophase I in the WT and m10690 mutant anthers.Anthers were harvested from the WT and m10690 mutant plants at different developmental stages. Gene expression was determined using qRT-PCR. OsACTIN1 was used as internal control. Data are shown as means ± SD (n = 3). *** indicates significance at P?≤?0.001 and ** indicates significance at P?≤?0.01 by student’s t test.

Fig. 5. Simultaneous identification of multiple causal mutations (SIMM) analysis of m10690 mutant and gene structure of OsRAD1.A, Mapping of the OsRAD1 gene based on the distribution of Euclidian Distance (ED6) values of SNPs along the rice chromosomes. B, OsRAD1 gene structure. The black boxes indicate exons, the gray boxes indicate 5′-UTR and 3′-UTR, the black lines indicate introns and the red arrows indicate the mutation sites in the original m10690 mutant and three other mutants generated by CRISPR knockout. The blue line represents the DNA fragment for transgenic complementation. C, Alignment of the C-terminal sequences of OsRAD1.1, OsRAD1.2, OsRAD1.3 and OsRAD1M.

Supplemental Fig. 3. Transgenic complementation of the m10690 mutant.(A) Panicle of the WT HHZ, m10690 mutant, and transgenic-complemented m10690 plants. (B-D) I2-KI stained pollen grains of the WT (B), m10690 mutant (C), and transgenic-complemented osrad1 plants (D). Scale bars: A, 2cm; B-D, 100μm.

Supplemental Fig. 4. The positions of the primers for RT-PCR.(A), OsRAD1-qPCR; (B), OsRAD1.1-qPCR; (C), OsRAD1.2+3-qPCR; (D), OsRAD1.2-qPCR; (E), OsRAD1.3-qPCR. The black boxes indicate exons, the gray boxes indicate 5’UTR and 3’UTR, and the black lines indicate introns.

Fig. 6. Analysis of expression patterns of OsRAD1. A, Expression levels of OsRAD1 at different tissues and anther developmental stages. B, Expression levels of OsRAD1.1 at different tissues and anther developmental stages. C, Expression levels of OsRAD1.2 plus OsRAD1.3 at different tissues and anther developmental stages. D, Expression levels of OsRAD1.1 transcripts in anthers and pistils at different developmental stages. E, Expression levels of OsRAD1.2 transcripts in anthers and pistils at different developmental stages. F, Expression levels of OsRAD1.3 transcripts in anthers and pistils at different developmental stages.St6 to St12 are different developmental stages of anthers. Pistils and other tissues in A?C were harvested from plants at the flowering stage. Anthers and pistils in D?F were collected at St6 to St12. OsACTIN1 was used as the internal control. Data are Mean ± SD (n = 3).

Fig. 7. Phenotypes of CRISPR mutant alleles and transgenic plants overexpressing OsRAD1.2 plus OsRAD1.3.A, Wuyungeng 7. B, cr-1. C, cr-2. D, cr-3. E, cr-2 overexpressing OsRAD1.2. F, cr-2 overexpressing OsRAD1.3. G, Wuyungeng 7 over- expressing OsRAD1.2. H, Wuyungeng 7 overexpressing OsRAD1.3. The seed-setting and pollen grains stained with I2-KI are shown. White scale bars, 5 cm. Red scale bars, 100 μm. I, Relative expression levels of OsRAD1.2 and OsRAD1.3 in nontransgenic (N) and transgenic (P) plants of Wuyungeng 7 (WT) and cr-2 backgrounds. OsACTIN1 was used as the internal control. Data are shown as Mean ± SD (n = 3).

Supplemental Fig. 5. Alignment of OsRAD1.1, OsRAD1.2, OsRAD1.3, and OsRAD1.1M with RAD1 proteins from other plant species.Protein sequences were retrieved from NCBI by BLASTP search using OsRAD1.1 as query. The unique C-terminal domain sequence of OsRAD1.1 is underlined.

Fig. 8. Yeast two-hybrid assay of OsRAD1.1, OsRAD1.2, OsRAD1.3 and OsRAD1M interaction with RAD9, HUS1 and RAD17.SD/-Leu-Trp was used to test the co-transformation efficiency. The interactions were verified by the growth of yeast on selective medium SD/-Ade-His-Leu-Trp. The interaction of AD-T7 and BD-53 was included as the positive control, the interaction of AD-T7 and BD-lam was included as the negative control, and the interaction of BD and AD empty vectors was also included as the negative control.

Supplemental Fig. 6. Alignment of the predicated steric structures of OsRAD1.1 with OsRAD1.2, OsRAD1.3, and OsRAD1M.

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