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Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA.

Yang X, Ren W, Zhao Q, Zhang P, Wu F, He Y - Nucleic Acids Res. (2014)

Bottom Line: Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants.HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA-dsRNA junction in pri-miRNA.These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

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Crucial role of Gly147 and Leu165 in HYL1 dimerization. (A) BiFC analysis showing homodimerization of HYL1 and HYL1 mutants. (B) Pull-down assay results showing protein–protein interaction of HYL1 mutants with themselves. T146E, HYL1T146E; G147E, HYL1G147E; I158E, HYL1I158E; L165E, HYL1L165E; L166E, HYL1L166E. (C) Pull-down assay results showing protein–protein interaction of HYL1 mutants with wild-type HYL1.
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Figure 2: Crucial role of Gly147 and Leu165 in HYL1 dimerization. (A) BiFC analysis showing homodimerization of HYL1 and HYL1 mutants. (B) Pull-down assay results showing protein–protein interaction of HYL1 mutants with themselves. T146E, HYL1T146E; G147E, HYL1G147E; I158E, HYL1I158E; L165E, HYL1L165E; L166E, HYL1L166E. (C) Pull-down assay results showing protein–protein interaction of HYL1 mutants with wild-type HYL1.

Mentions: Bimolecular fluorescence complementation (BiFC) was used to evaluate the effects of the point mutations on HYL1 dimerization. The same HYL1 mutants were fused with YFP at either the N- or C-terminus and introduced into the Arabidopsis protoplast for transient expression. The HYL1 mutants were visualized via confocal microscopy. Fluorescently labeled HYL1 mutants during the formation of BiFC complexes showed that the fluorescence intensity for HYL1G147E (G147E) or HYL1L165E (L165E) was significantly diminished, revealing that the homodimerization of these mutants are weakened (Figure 2A). By contrast, HYL1C122R (C122R), HYL1R130E (R130E), HYL1T146R (T146R), HYL1T146E (T146E), HYL1I158E (I158E) and HYL1L166E (L166E) showed almost the same fluorescence intensity as the wild-type HYL1 did, meaning that they also formed homodimers (Supplementary Figure S3A). Weaker fluorescence intensity was also found when G147E and L165E were cotransformed with wild-type HYL1. To exclude the possibility of transient transfer efficiency, western blotting was carried out to detect the mutated HYL1 fused with YFP. All five HYL1 mutants examined showed almost the same intensity of YFP (Supplementary Figure S4) in the protoplasts, demonstrating that the difference in transfer efficiency in the BiFC analysis was not significant. Therefore, we conclude that Gly147 and Leu165 are required for HYL1 dimerization. Using this method, we also examined the effects of the other three mutations (R151E, K154E and R162E) on their interaction with SE and DCL1, but found no difference from wild-type HYL1 (data not shown).


Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA.

Yang X, Ren W, Zhao Q, Zhang P, Wu F, He Y - Nucleic Acids Res. (2014)

Crucial role of Gly147 and Leu165 in HYL1 dimerization. (A) BiFC analysis showing homodimerization of HYL1 and HYL1 mutants. (B) Pull-down assay results showing protein–protein interaction of HYL1 mutants with themselves. T146E, HYL1T146E; G147E, HYL1G147E; I158E, HYL1I158E; L165E, HYL1L165E; L166E, HYL1L166E. (C) Pull-down assay results showing protein–protein interaction of HYL1 mutants with wild-type HYL1.
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Figure 2: Crucial role of Gly147 and Leu165 in HYL1 dimerization. (A) BiFC analysis showing homodimerization of HYL1 and HYL1 mutants. (B) Pull-down assay results showing protein–protein interaction of HYL1 mutants with themselves. T146E, HYL1T146E; G147E, HYL1G147E; I158E, HYL1I158E; L165E, HYL1L165E; L166E, HYL1L166E. (C) Pull-down assay results showing protein–protein interaction of HYL1 mutants with wild-type HYL1.
Mentions: Bimolecular fluorescence complementation (BiFC) was used to evaluate the effects of the point mutations on HYL1 dimerization. The same HYL1 mutants were fused with YFP at either the N- or C-terminus and introduced into the Arabidopsis protoplast for transient expression. The HYL1 mutants were visualized via confocal microscopy. Fluorescently labeled HYL1 mutants during the formation of BiFC complexes showed that the fluorescence intensity for HYL1G147E (G147E) or HYL1L165E (L165E) was significantly diminished, revealing that the homodimerization of these mutants are weakened (Figure 2A). By contrast, HYL1C122R (C122R), HYL1R130E (R130E), HYL1T146R (T146R), HYL1T146E (T146E), HYL1I158E (I158E) and HYL1L166E (L166E) showed almost the same fluorescence intensity as the wild-type HYL1 did, meaning that they also formed homodimers (Supplementary Figure S3A). Weaker fluorescence intensity was also found when G147E and L165E were cotransformed with wild-type HYL1. To exclude the possibility of transient transfer efficiency, western blotting was carried out to detect the mutated HYL1 fused with YFP. All five HYL1 mutants examined showed almost the same intensity of YFP (Supplementary Figure S4) in the protoplasts, demonstrating that the difference in transfer efficiency in the BiFC analysis was not significant. Therefore, we conclude that Gly147 and Leu165 are required for HYL1 dimerization. Using this method, we also examined the effects of the other three mutations (R151E, K154E and R162E) on their interaction with SE and DCL1, but found no difference from wild-type HYL1 (data not shown).

Bottom Line: Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants.HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA-dsRNA junction in pri-miRNA.These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

Show MeSH