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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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Homodimerization of HYL1 through dsRBD2. (A) Diagram of functional domains in HYL1 protein. dsRBD1, double-stranded RNA-binding domain 1; dsRBD2, double-stranded RNA-binding domain 2; NLS, nuclear localization signal; PPI, protein–protein interaction domain. (B) Homodimerization of HYL1 and HYL1 mutants in yeast two-hybrid assay. D1: dsRBD1; D2: dsRBD2; AD, GAL4 activation domain fusions; DB, GAL4 DNA binding domain fusions; -LT, medium without leucine and tryptophan; -LTAH, without leucine, tryptophan, adenine and histidine. Growth in the -LTAH medium indicates protein–protein interaction. (C) Pull-down assay results for HYL1 homodimers. D1, dsRBD1; D2, dsRBD2.
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Figure 1: Homodimerization of HYL1 through dsRBD2. (A) Diagram of functional domains in HYL1 protein. dsRBD1, double-stranded RNA-binding domain 1; dsRBD2, double-stranded RNA-binding domain 2; NLS, nuclear localization signal; PPI, protein–protein interaction domain. (B) Homodimerization of HYL1 and HYL1 mutants in yeast two-hybrid assay. D1: dsRBD1; D2: dsRBD2; AD, GAL4 activation domain fusions; DB, GAL4 DNA binding domain fusions; -LT, medium without leucine and tryptophan; -LTAH, without leucine, tryptophan, adenine and histidine. Growth in the -LTAH medium indicates protein–protein interaction. (C) Pull-down assay results for HYL1 homodimers. D1, dsRBD1; D2, dsRBD2.

Mentions: The two dsRBDs, putative protein–protein interaction domain and NLS of HYL1 are indicated in Figure 1A. To determine whether HYL1 forms a homodimer in vitro, we used full-length and truncated HYL1 for a yeast two-hybrid assay. We observed a direct interaction between two HYL1 molecules (Figure 1B). The dsRBD2 fragment interacted with full-length HYL1, whereas the dsRBD1 fragment did not. Moreover, one dsRBD2 fragment interacted with another dsRBD2 but not with dsRBD1. These findings indicate that HYL1 forms a homodimer through the dsRBD2 domains.


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)

Homodimerization of HYL1 through dsRBD2. (A) Diagram of functional domains in HYL1 protein. dsRBD1, double-stranded RNA-binding domain 1; dsRBD2, double-stranded RNA-binding domain 2; NLS, nuclear localization signal; PPI, protein–protein interaction domain. (B) Homodimerization of HYL1 and HYL1 mutants in yeast two-hybrid assay. D1: dsRBD1; D2: dsRBD2; AD, GAL4 activation domain fusions; DB, GAL4 DNA binding domain fusions; -LT, medium without leucine and tryptophan; -LTAH, without leucine, tryptophan, adenine and histidine. Growth in the -LTAH medium indicates protein–protein interaction. (C) Pull-down assay results for HYL1 homodimers. D1, dsRBD1; D2, dsRBD2.
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Figure 1: Homodimerization of HYL1 through dsRBD2. (A) Diagram of functional domains in HYL1 protein. dsRBD1, double-stranded RNA-binding domain 1; dsRBD2, double-stranded RNA-binding domain 2; NLS, nuclear localization signal; PPI, protein–protein interaction domain. (B) Homodimerization of HYL1 and HYL1 mutants in yeast two-hybrid assay. D1: dsRBD1; D2: dsRBD2; AD, GAL4 activation domain fusions; DB, GAL4 DNA binding domain fusions; -LT, medium without leucine and tryptophan; -LTAH, without leucine, tryptophan, adenine and histidine. Growth in the -LTAH medium indicates protein–protein interaction. (C) Pull-down assay results for HYL1 homodimers. D1, dsRBD1; D2, dsRBD2.
Mentions: The two dsRBDs, putative protein–protein interaction domain and NLS of HYL1 are indicated in Figure 1A. To determine whether HYL1 forms a homodimer in vitro, we used full-length and truncated HYL1 for a yeast two-hybrid assay. We observed a direct interaction between two HYL1 molecules (Figure 1B). The dsRBD2 fragment interacted with full-length HYL1, whereas the dsRBD1 fragment did not. Moreover, one dsRBD2 fragment interacted with another dsRBD2 but not with dsRBD1. These findings indicate that HYL1 forms a homodimer through the dsRBD2 domains.

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