Limits...
The Roles of Arabidopsis CDF2 in Transcriptional and Posttranscriptional Regulation of Primary MicroRNAs.

Sun Z, Guo T, Liu Y, Liu Q, Fang Y - PLoS Genet. (2015)

Bottom Line: CDF2 binds directly to the promoters of some miRNAs and works as a transcription activator or repressor for these miRNA genes.CDF2 binds preferentially to the pri-miRNAs regulated by itself and affects DCL1-mediated processing of these pri-miRNAs.We conclude that CDF2 regulates a group of pri-miRNAs at both the transcriptional and posttranscriptional levels to maintain proper levels of their mature miRNAs to control 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, China.

ABSTRACT
The precise regulation of microRNA (miRNA) transcription and processing is important for eukaryotic development. Plant miRNAs are first transcribed as stem-loop primary miRNAs (pri-miRNAs) by RNA polymerase II,then cleaved in the nucleus into mature miRNAs by Dicer-like 1 (DCL1). We identified a cycling DOF transcription factor, CDF2, which interacts with DCL1 and regulates the accumulation of a population of miRNAs. CDF2 binds directly to the promoters of some miRNAs and works as a transcription activator or repressor for these miRNA genes. CDF2 binds preferentially to the pri-miRNAs regulated by itself and affects DCL1-mediated processing of these pri-miRNAs. Genetically, CDF2 works in the same pathway as miR156 or miR172 to control flowering. We conclude that CDF2 regulates a group of pri-miRNAs at both the transcriptional and posttranscriptional levels to maintain proper levels of their mature miRNAs to control plant development.

No MeSH data available.


CDF2 interacts with DCL1.(A) Yeast two hybrid assays show the interactions between CDF2 and DCL1-RBD or HYL1. Co-transformed yeast colonies were spotted on the selective SC medium minus Trp and Leu, and then grown on SC medium minus His, Trp, and Leu supplemented with 5mM 3-amino-1, 2, 4-triazole (3-AT). (B) GST and MBP pull-down assays show the interaction between CDF2 and DCL1. (C) Co-IP assay shows the interaction between CDF2 and DCL1. The protein extracts from 22-day-old Arabidopsis plants coexpressing pCDF2::CDF2-HA and pDCL1::DCL1-YFP were incubated with anti-HA–conjugated agarose. The pellet was analyzed by immunoblotting with anti-HA and anti-GFP antibodies. (D) Bimolecular Fluorescence Complementation (BiFC) assays show that CDF2 or the C-terminal fragment of CDF2 (CDF2-C) interact with DCL1/HYL1 in D-bodies, while no interactions were observed between CDF2 and DCL1-9 or the N-terminal fragment of CDF2 (CDF2-N) and DCL1. Scale bar = 10μm. (E) Yeast two hybrid assays show that the C terminal fragment of CDF2 interacts with DCL1-RBD. C1, aa 189–457; C2, aa 271–457; C3, aa 360–457.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608766&req=5

pgen.1005598.g001: CDF2 interacts with DCL1.(A) Yeast two hybrid assays show the interactions between CDF2 and DCL1-RBD or HYL1. Co-transformed yeast colonies were spotted on the selective SC medium minus Trp and Leu, and then grown on SC medium minus His, Trp, and Leu supplemented with 5mM 3-amino-1, 2, 4-triazole (3-AT). (B) GST and MBP pull-down assays show the interaction between CDF2 and DCL1. (C) Co-IP assay shows the interaction between CDF2 and DCL1. The protein extracts from 22-day-old Arabidopsis plants coexpressing pCDF2::CDF2-HA and pDCL1::DCL1-YFP were incubated with anti-HA–conjugated agarose. The pellet was analyzed by immunoblotting with anti-HA and anti-GFP antibodies. (D) Bimolecular Fluorescence Complementation (BiFC) assays show that CDF2 or the C-terminal fragment of CDF2 (CDF2-C) interact with DCL1/HYL1 in D-bodies, while no interactions were observed between CDF2 and DCL1-9 or the N-terminal fragment of CDF2 (CDF2-N) and DCL1. Scale bar = 10μm. (E) Yeast two hybrid assays show that the C terminal fragment of CDF2 interacts with DCL1-RBD. C1, aa 189–457; C2, aa 271–457; C3, aa 360–457.

Mentions: To identify new components involved in regulation of miRNA biogenesis, we performed a yeast two-hybrid screening for proteins that interact with the two C-terminal dsRBDs of DCL1 (DCL1-RBD, aa 1733–1910), which complement the phenotypes of hyl1 mutant and are important for protein–protein interactions and pri-miRNA bindings [24, 25]. Among the obtained 54 independent prey clones, 15 represented DCL1, which is consistent with the findings of previous studies that DCL1 can interact with itself [24,25]. Four independent prey clones contained various C-terminal fragments of CDF2. We then examined the interactions between full length of DCL1/HYL1 and CDF2 by yeast two-hybrid assays, the results showed that CDF2 can interact with DCL1 and HYL1 (Fig 1A).


The Roles of Arabidopsis CDF2 in Transcriptional and Posttranscriptional Regulation of Primary MicroRNAs.

Sun Z, Guo T, Liu Y, Liu Q, Fang Y - PLoS Genet. (2015)

CDF2 interacts with DCL1.(A) Yeast two hybrid assays show the interactions between CDF2 and DCL1-RBD or HYL1. Co-transformed yeast colonies were spotted on the selective SC medium minus Trp and Leu, and then grown on SC medium minus His, Trp, and Leu supplemented with 5mM 3-amino-1, 2, 4-triazole (3-AT). (B) GST and MBP pull-down assays show the interaction between CDF2 and DCL1. (C) Co-IP assay shows the interaction between CDF2 and DCL1. The protein extracts from 22-day-old Arabidopsis plants coexpressing pCDF2::CDF2-HA and pDCL1::DCL1-YFP were incubated with anti-HA–conjugated agarose. The pellet was analyzed by immunoblotting with anti-HA and anti-GFP antibodies. (D) Bimolecular Fluorescence Complementation (BiFC) assays show that CDF2 or the C-terminal fragment of CDF2 (CDF2-C) interact with DCL1/HYL1 in D-bodies, while no interactions were observed between CDF2 and DCL1-9 or the N-terminal fragment of CDF2 (CDF2-N) and DCL1. Scale bar = 10μm. (E) Yeast two hybrid assays show that the C terminal fragment of CDF2 interacts with DCL1-RBD. C1, aa 189–457; C2, aa 271–457; C3, aa 360–457.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608766&req=5

pgen.1005598.g001: CDF2 interacts with DCL1.(A) Yeast two hybrid assays show the interactions between CDF2 and DCL1-RBD or HYL1. Co-transformed yeast colonies were spotted on the selective SC medium minus Trp and Leu, and then grown on SC medium minus His, Trp, and Leu supplemented with 5mM 3-amino-1, 2, 4-triazole (3-AT). (B) GST and MBP pull-down assays show the interaction between CDF2 and DCL1. (C) Co-IP assay shows the interaction between CDF2 and DCL1. The protein extracts from 22-day-old Arabidopsis plants coexpressing pCDF2::CDF2-HA and pDCL1::DCL1-YFP were incubated with anti-HA–conjugated agarose. The pellet was analyzed by immunoblotting with anti-HA and anti-GFP antibodies. (D) Bimolecular Fluorescence Complementation (BiFC) assays show that CDF2 or the C-terminal fragment of CDF2 (CDF2-C) interact with DCL1/HYL1 in D-bodies, while no interactions were observed between CDF2 and DCL1-9 or the N-terminal fragment of CDF2 (CDF2-N) and DCL1. Scale bar = 10μm. (E) Yeast two hybrid assays show that the C terminal fragment of CDF2 interacts with DCL1-RBD. C1, aa 189–457; C2, aa 271–457; C3, aa 360–457.
Mentions: To identify new components involved in regulation of miRNA biogenesis, we performed a yeast two-hybrid screening for proteins that interact with the two C-terminal dsRBDs of DCL1 (DCL1-RBD, aa 1733–1910), which complement the phenotypes of hyl1 mutant and are important for protein–protein interactions and pri-miRNA bindings [24, 25]. Among the obtained 54 independent prey clones, 15 represented DCL1, which is consistent with the findings of previous studies that DCL1 can interact with itself [24,25]. Four independent prey clones contained various C-terminal fragments of CDF2. We then examined the interactions between full length of DCL1/HYL1 and CDF2 by yeast two-hybrid assays, the results showed that CDF2 can interact with DCL1 and HYL1 (Fig 1A).

Bottom Line: CDF2 binds directly to the promoters of some miRNAs and works as a transcription activator or repressor for these miRNA genes.CDF2 binds preferentially to the pri-miRNAs regulated by itself and affects DCL1-mediated processing of these pri-miRNAs.We conclude that CDF2 regulates a group of pri-miRNAs at both the transcriptional and posttranscriptional levels to maintain proper levels of their mature miRNAs to control 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, China.

ABSTRACT
The precise regulation of microRNA (miRNA) transcription and processing is important for eukaryotic development. Plant miRNAs are first transcribed as stem-loop primary miRNAs (pri-miRNAs) by RNA polymerase II,then cleaved in the nucleus into mature miRNAs by Dicer-like 1 (DCL1). We identified a cycling DOF transcription factor, CDF2, which interacts with DCL1 and regulates the accumulation of a population of miRNAs. CDF2 binds directly to the promoters of some miRNAs and works as a transcription activator or repressor for these miRNA genes. CDF2 binds preferentially to the pri-miRNAs regulated by itself and affects DCL1-mediated processing of these pri-miRNAs. Genetically, CDF2 works in the same pathway as miR156 or miR172 to control flowering. We conclude that CDF2 regulates a group of pri-miRNAs at both the transcriptional and posttranscriptional levels to maintain proper levels of their mature miRNAs to control plant development.

No MeSH data available.