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Similar to Sequestration of a Transposon-Derived siRNA by an Imprinted Gene Induces Reproductive Isolation
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Sequestration of a Transposon-Derived siRNA by an Imprinted Gene Induces Reproductive Isolation
- 1. Sequestration of a Transposon- Derived siRNA by a Target Mimic Imprinted Gene Induces Post-zygotic Reproductive Isolation in Arabidopsis Presented by Nitika ,Kajol B.M Singh , Vijay and Rajan (M.sc. Botany(F))
- 2. BACKGROUND Genomic imprinting is an epigenetic phenomenon occurring in mammals and flowering plants that leads to parent-of-origin specific differential expression of maternally and paternally inherited alleles. maternally expressed gene and paternally silent are referred to as MEGs (maternally expressed imprinted genes paternally expressed and maternally silent are referred to as PEGs (paternally expressed imprinted genes). Wolff, P., Jiang, H., Wang, G., Santos-Gonzalez, J., & Köhler, C. (2015). Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana. Elife, 4, e10074.
- 3. During reproduction, plant genomes undergo significant reconfiguration of chromatin structure and DNA methylation HOW IMPRINTING HAPPEN? vegetative cell undergoes substantial DNA demethylation due to reduced expression of DECREASE in DNA METHYLATION1 (DDM1) and activity of the DEMETER (DME) demethylase that frequently targets TEs During female gametophyte development, active demethylation by DME occurs throughout the genome of the central cell IMPRINTING SETTING PRIOR TO FERTILIZATION Gehring, M. (2013). Genomic imprinting: parental lessons from plants. Annu Rev Genet, 47, 187-208. Lessons From Plants, Review in Advance
- 4. Imprinting effect is primarily restricted to the endosperm models that integrate known chromatin dynamics during reproduction to explain how the insertion of a TE could lead to a MEG or PEG imprinting status for a gene. Ref. Sarah N Anderson and Nathan M Springer, Potential roles for transposable elements in creating imprinted expression, Current Opinion in Genetics & Development 2018, 49:8–14
- 5. GENOMIC IMPRINTING AND TRIPLOID BLOCK fusion of the maternal homodiploid central cell with a paternal haploid sperm cell, the expected ratio of maternal to paternal transcripts is 2:1 endosperm balance number changes in the ratio of maternal and paternal genomes Unbalanced expression of imprinted genes consequences for endosperm development, leading to seed collapse Lafon‐Placette, C., & Köhler, C. (2016). Endosperm‐based postzygotic hybridization barriers: developmental mechanisms and evolutionary drivers. Molecular Ecology, 25(11), 2620-2629.
- 6. paternally expressed imprinted gene PEG2 transcript sequesters the transposable element (TE)-derived small interfering RNA (siRNA) siRNA854 in the endosperm reproductive barrier and prevents successful hybridizations between plants differing in chromosome number (ploidy) Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.
- 7. Hybridizations of diploid (2x) and tetraploid (4x) Arabidopsis plants cause strongly increased expression of paternally expressed imprinted genes (PEGs) ADMETOS (ADM), SUVH7, PEG2, and PICKLE RELATED2 share a common mode of action, acts as a dosage-sensitive regulator of triploid seed development and increased ADM expression in triploid seeds causes endosperm cellularization failure leading to seed abortion functions only in syncytial endosperm Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.
- 8. In Arabidopsis, at time of microspore to form the pollen grain, the vegetative cell undergoes substantial DNA demethylation due activity of the DEMETER (DME) demethylase that frequently targets Transposable element. The de-repression of TEs in the vegetative cell is followed by generation of epigenetically activated small interfering RNAs (easiRNAs) increased levels of easiRNAs negatively correlate with DNA methylation established by the RNA- dependent DNA methylation (RdDM) pathway suggesting a dose- dependent role of easiRNAs in controlling RdDM activity Furthermore, depletion of easiRNAs strongly reduces PEG expression and restores viability of triploid seeds indicating that easiRNAs act upstream in controlling PEG expression Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.
- 9. In this work, they show that transposable element (TE)-derived small interfering RNA (siRNA) siRNA854 present in the vegetative cell of pollen then transferred to the central cell of the female gametophyte after fertilization , where it is captured by PEG2 This sequestering inhibits the targeting of siRNA854 natural target genes like UBP1b Sequestration of siRNA854 as a consequence of increased PEG2 transcript levels is causally responsible for triploid seed inviability. Thus, the balance of a male gamete accumulating TE-derived siRNA and a PEG regulates triploid seed viability, revealing a trans generational speciation mechanism . Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.
- 10. RESULTS PEG2 Is Transcribed, but Not Translated, in the Endosperm PEG2 Translation in the Endosperm Is Impaired by siRNA854 The siRNA854 Binding Site Is Essential to Cause Triploid Seed Abortion The PEG2 mRNA Rather Than the PEG2 Protein Is Functionally Relevant in the Triploid Block PEG2 Functions as an siRNA Sponge after Fertilization in the Endosperm Regulation of UBP1b by siRNA854 Is Causally Connected to Triploid Seed Viability siRNA854 Is Transferred to the Central Cell via Sperm Cells
- 11. Experimental Models Arabidopsis thaliana Columbia (Col-0) ubp1b-3 mutant Peg2 mutant osd1-1 and osd1-3 mutants T-DNA Insertion lines, PCR confirmed For crossing experiments, designated female plants were emasculated, and the pistils were manually pollinated 2 days after emasculation. Transgenic lines Floral Dip method A. Thaliana Seeds Stratification for 2 days at 4C Surface sterilised(5% sodium hypochlorite and 0.01% Triton X-100) Plants transferred into soil and grown in a growth chamber Germination on half strength MS medium (1% sucrose) 16 h L/8 h D, 21C
- 12. Vectors Fusion Vector Over-expression Vector
- 13. Translational reporter PEG2::PEG2-GFP Transcriptional reporter PEG2::H2B-GFP Silent mutant of PEG2 (PEG2::PEG2MUT-GFP) Frame-shift PEG2 (RPS5A::PEG2FS) Truncated PEG2 (RPS5A::PEG2TR) Tissue-specific PEG2 constructs (PHE1::PEG2 & MGH3::PEG2) Mutant PEG2 with full sequence complementarity to siRNA854 (PEG2::PEG2PM- GFP) 35S::PEG2-GFP PEG2 GFP PEG2 H2B GFP PEG2 PEG2FS GFP RPS5A PEG2MUT GFP PEG2 PEG2 PHE1 PEG2TR GFP RPS5A PEG2 MGH3 PEG2PM GFP PEG2 PEG2 GFP 35S C O N S T R U C T S
- 14. Triploid seeds generated from pollinating wild-type plants with diploid pollen of the omission of second division 1 (osd1) mutant abort at high frequency, while osd1 peg2 double mutant pollen gives rise to viable triploid progeny. . PEG2 Is Transcribed, but Not Translated, in the Endosperm PEG2 (AT1G49290) encodes for an unknown protein with no predicted functional domains (Pollen 2n) (Pollen 2n)
- 15. Localized PEG2 using a translational reporter construct of the PEG2 promoter fused to the PEG2 coding region and the green fluorescent protein GFP; PEG2::PEG2-GFP Monitored GFP fluorescence and found a strong signal surrounding the sperm nuclei of pollen. The presence of PEG2::PEG2-GFP in osd1 peg2 restored triploid seed abortion, revealed that this construct is functional and can complement the peg2 mutant. Sperm cell GPF Signal surrounding nuclei of sperm cell
- 16. No GFP fluorescence in diploid or triploid seeds generated from pollinations with haploid or diploid PEG2:: PEG2-GFP pollen. This was in sharp contrast to the strong increase of the PEG2 transcript levels in triploid seeds, indicating that these transcripts accumulate but fail to be translated. Transcripts derived from PEG2::PEG2-GFP accumulated to similar levels as those of the endogenous PEG2 gene A transcriptional reporter for PEG2 containing the PEG2 promoter sequence driving a nuclear-localized histone H2B-GFP fusion was highly active in sperm cells and in the endosperm Seed without GFP Signal at 4 days after polliantion
- 17. PEG2 Translation in the Endosperm Is Impaired by siRNA854 Queried PEG2 sequence for possible microRNA (miRNA) target sites 5 members of the miRNA854/siRNA854 family predicted to target the PEG2 cDNA sequence at nucleotides 614 to 634. These mismatches indicate a potential destabilization of the siRNA854 cleavage at its central position that leads to translational repression. Target site contained a mismatch at position 10 and two non-Watson-Crick base interactions at positions 9 and 11 of the siRNA/PEG2 mRNA hybrid. Introduced three silent mutations into the siRNA854 binding site, to test whether it is responsible for PEG2 translational inhibition Mismatch and non watson- crick base interaction
- 18. Showed GFP fluorescence detected surrounding sperm nuclei when expressed mutated version of PEG2 (PEG2MUT), similar to the signal detected when expressing the non- mutated version of PEG2. In contrast to the non-mutated version, this construct was translated in the endosperm, as evidenced by a detectable GFP signal surrounding the endosperm nuclei
- 19. Consistently, the PEG2MUT-GFP protein was detected by immunoblot analysis, while no signal was detected in seeds expressing PEG2-GFP . Jointly, these results strongly support that PEG2 translation in the endosperm is impaired by siRNA854.
- 20. • Introduced the two versions of PEG2 with the natural and mutated siRNA854 binding sites into the osd1 peg2 double mutant and tested viability of the progeny obtained from crossing these plants to a diploid maternal plant. • In contrast to the complete complementation of the abortion phenotype when expressing PEG2::PEG2-GFP, PEG2::PEG2MUT-GFP was not able to confer full complementation . • While the decrease in viability of triploid peg2; PEG2::PEG2MUT-GFP seeds was significant compared to triploid peg2 seeds, the difference was rather minor and differed drastically from the complete suppression of viability conferred by expressing PEG2::PEG2-GFP. The siRNA854 Binding Site Is Essential to Cause Triploid Seed Abortion
- 22. So, this study says that PEG serves as sponge for epigenetically controlled siRNA (produced in the pollen grain).Dosage of siRNA854 in endosperm is responsible for triploid seed survival. Its already know ,Coding and non coding RNAs can act as molecular sponge to regulate abundance of miRNAs (Franco- Zorilla et al. 2007) According to Author, No one before this paper reported about molecular sponge for TE-Derived siRNA. But this paper does! So know we know that dosage of TE- derived siRNA854 work as a reproductive barrier b/w plants with different ploidy. As per Borges et al., It is know that easiRNA is actually responsible for triploid block by affecting the imprinted genes, and offcourse siRNA is a member of easiRNA (LTR retrotransposons).These LTR retrotransposons activity depends on various DCL 2,4 etc. From above points we can say that, dependence of PEG2 to deplete siRNA854 depends on easiRNA.
- 23. The possible explanation for the paradox is that reduced dosage of easiRNA in triploid seeds restores wild type levels of PEG2 and other PEG such as ADM in the intro portion . They are responsible for seed abortion ,but mutation in ADM leads to viable seed formation.
- 24. Model Explaining the Role of PEG2 in Establishing Interploidy Seed Abortion -In pollen , PEG2 is expressed in sperm cells. -The siRNA854 is present in the vegetative cell, and transported to sperm cells. -In sperm,siRNA854 doesn’t efficiently inhibit translation of PEG2, and PEG2 surrounds sperm nuclei .
- 26. INFERENCES Normal fertilisation. PEG2 highly regulated ,cause depletion of siRNA854. Hence , reduction in PEGs expression ,bypasses the need of siRNA854 and restores triploid seed viability.