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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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Deficiency of the hyl1-3 allele in miRNA biogenesis. (A) RT-PCR results showing expression of endogenous G147E in the hyl1-3 mutants. (B) Western blotting results showing endogenous G147E protein in the hyl1-3 mutants. (C) Northern blotting results showing miRNA accumulation in the hyl1-3 mutants. (D) Real-time PCR results showing the expression levels of some miRNA-targeted genes. (E) Real-time PCR results showing the accumulation levels of pri-miRNAs. (F) RT-PCR results showing pri-miRNA abundances in immunoprecipitates of Col and hyl1-3 plants.
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Figure 6: Deficiency of the hyl1-3 allele in miRNA biogenesis. (A) RT-PCR results showing expression of endogenous G147E in the hyl1-3 mutants. (B) Western blotting results showing endogenous G147E protein in the hyl1-3 mutants. (C) Northern blotting results showing miRNA accumulation in the hyl1-3 mutants. (D) Real-time PCR results showing the expression levels of some miRNA-targeted genes. (E) Real-time PCR results showing the accumulation levels of pri-miRNAs. (F) RT-PCR results showing pri-miRNA abundances in immunoprecipitates of Col and hyl1-3 plants.

Mentions: To examine whether the endogenous G147E in the hyl1-3 plants influences homodimerization as exogenous G147E, we used HYL1 antibody to detect the possible dimerization of G147E in the extracts of Arabidopsis under native condition. As expected, we were able to detect HYL1 homodimer in the wild-type and hyl1-2;I158E plants. (Figure 5C). In the hyl1-3 plants, HYL1 monomer rather than homodimer was detected. This result indicated that endogenous G147E in the hyl1-3 plants impaired homodimerization in vivo. In the hyl1-3 mutants, the expression levels of HYL1 gene and HYL1 protein were not different from the wild type (Figure 6A and B); the accumulation of the miRNAs examined was much less than that in wild type, though it was slightly higher than that in hyl1-2 (Figure 6C), and the genes targeted by these miRNAs were up-regulated (FigureĀ 6D). These results suggest that impairment of HYL1 dimerization affects the biological function of HYL1 in miRNA biogenesis and miRNA-guided gene silencing.


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)

Deficiency of the hyl1-3 allele in miRNA biogenesis. (A) RT-PCR results showing expression of endogenous G147E in the hyl1-3 mutants. (B) Western blotting results showing endogenous G147E protein in the hyl1-3 mutants. (C) Northern blotting results showing miRNA accumulation in the hyl1-3 mutants. (D) Real-time PCR results showing the expression levels of some miRNA-targeted genes. (E) Real-time PCR results showing the accumulation levels of pri-miRNAs. (F) RT-PCR results showing pri-miRNA abundances in immunoprecipitates of Col and hyl1-3 plants.
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Related In: Results  -  Collection

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Figure 6: Deficiency of the hyl1-3 allele in miRNA biogenesis. (A) RT-PCR results showing expression of endogenous G147E in the hyl1-3 mutants. (B) Western blotting results showing endogenous G147E protein in the hyl1-3 mutants. (C) Northern blotting results showing miRNA accumulation in the hyl1-3 mutants. (D) Real-time PCR results showing the expression levels of some miRNA-targeted genes. (E) Real-time PCR results showing the accumulation levels of pri-miRNAs. (F) RT-PCR results showing pri-miRNA abundances in immunoprecipitates of Col and hyl1-3 plants.
Mentions: To examine whether the endogenous G147E in the hyl1-3 plants influences homodimerization as exogenous G147E, we used HYL1 antibody to detect the possible dimerization of G147E in the extracts of Arabidopsis under native condition. As expected, we were able to detect HYL1 homodimer in the wild-type and hyl1-2;I158E plants. (Figure 5C). In the hyl1-3 plants, HYL1 monomer rather than homodimer was detected. This result indicated that endogenous G147E in the hyl1-3 plants impaired homodimerization in vivo. In the hyl1-3 mutants, the expression levels of HYL1 gene and HYL1 protein were not different from the wild type (Figure 6A and B); the accumulation of the miRNAs examined was much less than that in wild type, though it was slightly higher than that in hyl1-2 (Figure 6C), and the genes targeted by these miRNAs were up-regulated (FigureĀ 6D). These results suggest that impairment of HYL1 dimerization affects the biological function of HYL1 in miRNA biogenesis and miRNA-guided gene silencing.

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