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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.


Related in: MedlinePlus

CDF2 binds to pri-miRNAs and affects the binding of DCL1 to pri-miRNAs.(A) RNA EMSAs show the effect of CDF2 on the binding of DCL1 to pri-miRNA167. (B) An RIP assay was performed using 22-day-old plants, RNA fragments were immunoprecipitated with the GFP antibody and subsequently subjected to qRT-PCR analysisfor the indicated pri-miRNAs. Data are given as means ± SD (n = 3).
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pgen.1005598.g004: CDF2 binds to pri-miRNAs and affects the binding of DCL1 to pri-miRNAs.(A) RNA EMSAs show the effect of CDF2 on the binding of DCL1 to pri-miRNA167. (B) An RIP assay was performed using 22-day-old plants, RNA fragments were immunoprecipitated with the GFP antibody and subsequently subjected to qRT-PCR analysisfor the indicated pri-miRNAs. Data are given as means ± SD (n = 3).

Mentions: To investigate the biological role of the interaction between CDF2 and DCL1 in miRNA biogenesis, we performed RNA competitive electrophoretic mobility shift assays to test whether CDF2 affects the well-known binding activity of DCL1-RBD to pri-miRNAs. The reactions were performed using a fixed DCL1-RBD concentration and increasing amounts of CDF2. Interestingly, similar to DCL1-RBD (Figs 4A, lane1, and S7A–S7D, lane1), CDF2 was also observed to bind to pri-miR167b (Figs 4A, lane2, and S7A-S7D, lane2), and the binding ability of DCL1-RBD to the pri-miRNA decreased as the concentration of CDF2 increased (Figs 4A and S7A–S7D, lane 3–7), indicating that the interaction between CDF2 and DCL1-RBD affects the binding of DCL1-RBD to the pri-miRNA in vitro. In addition, we also found that the binding of CDF2 to pri-miRNAs was mainly mediated by its C-terminal fragment (S7E Fig). To test the pri-miRNA binding of CDF2 in vivo, an RNA immunoprecipitation assay was performed. Using a GFP antibody, we immunoprecipitated CDF2-YFP and DCL1-YFP complexes from the 22-day-old seedlings of pCDF2::CDF2-YFP/Col (S2A Fig), pDCL1::DCL1-YFP/Col, pDCL1::DCL1-YFP/cdf2, and pDCL1::DCL1-YFP/Col X p35S::CDF2-HA/Col line which was generated by crossing pDCL1::DCL1-YFP with p35S::CDF2-HA transgenic lines, using Col as a negative control of plants. In parallel, an IgG antibody- immunoprecipitated samples from these lines were used as a negative control of the antibody. Interestingly, the immunoprecipitated CDF2-YFP complex (S8 Fig) contains pri-miR156a, pri-miR172b, pri-miR319b, and pri-miR167b, which have altered levels in the cdf2 mutant, but not pri-miR164a, which showed no change in cdf2 mutant (Fig 4B, S1 Table). In contrast, DCL1 was able to bind to all 5 pri-miRNAs examined (Fig 4B). The bindings of the DCL1 to pri-miRNAs increase in the pDCL1::DCL1-YFP/cdf2 and decrease in the line of pDCL1::DCL1-YFP/Col X p35S::CDF2-HA/Col. These results suggested that CDF2 binds mainly to the CDF2-regulated pri-miRNAs in vivo, possibly due to that CDF2 has more accessibility to these pri-miRNAs transcribed with the aid of the transcription complex containing CDF2, this was supported by a recent report that transcription and processing of primary microRNAs are coupled by Elongator complex [30], resulting in that CDF2 only affects the processing of a subset of pri-miRNAs.


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 binds to pri-miRNAs and affects the binding of DCL1 to pri-miRNAs.(A) RNA EMSAs show the effect of CDF2 on the binding of DCL1 to pri-miRNA167. (B) An RIP assay was performed using 22-day-old plants, RNA fragments were immunoprecipitated with the GFP antibody and subsequently subjected to qRT-PCR analysisfor the indicated pri-miRNAs. Data are given as means ± SD (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608766&req=5

pgen.1005598.g004: CDF2 binds to pri-miRNAs and affects the binding of DCL1 to pri-miRNAs.(A) RNA EMSAs show the effect of CDF2 on the binding of DCL1 to pri-miRNA167. (B) An RIP assay was performed using 22-day-old plants, RNA fragments were immunoprecipitated with the GFP antibody and subsequently subjected to qRT-PCR analysisfor the indicated pri-miRNAs. Data are given as means ± SD (n = 3).
Mentions: To investigate the biological role of the interaction between CDF2 and DCL1 in miRNA biogenesis, we performed RNA competitive electrophoretic mobility shift assays to test whether CDF2 affects the well-known binding activity of DCL1-RBD to pri-miRNAs. The reactions were performed using a fixed DCL1-RBD concentration and increasing amounts of CDF2. Interestingly, similar to DCL1-RBD (Figs 4A, lane1, and S7A–S7D, lane1), CDF2 was also observed to bind to pri-miR167b (Figs 4A, lane2, and S7A-S7D, lane2), and the binding ability of DCL1-RBD to the pri-miRNA decreased as the concentration of CDF2 increased (Figs 4A and S7A–S7D, lane 3–7), indicating that the interaction between CDF2 and DCL1-RBD affects the binding of DCL1-RBD to the pri-miRNA in vitro. In addition, we also found that the binding of CDF2 to pri-miRNAs was mainly mediated by its C-terminal fragment (S7E Fig). To test the pri-miRNA binding of CDF2 in vivo, an RNA immunoprecipitation assay was performed. Using a GFP antibody, we immunoprecipitated CDF2-YFP and DCL1-YFP complexes from the 22-day-old seedlings of pCDF2::CDF2-YFP/Col (S2A Fig), pDCL1::DCL1-YFP/Col, pDCL1::DCL1-YFP/cdf2, and pDCL1::DCL1-YFP/Col X p35S::CDF2-HA/Col line which was generated by crossing pDCL1::DCL1-YFP with p35S::CDF2-HA transgenic lines, using Col as a negative control of plants. In parallel, an IgG antibody- immunoprecipitated samples from these lines were used as a negative control of the antibody. Interestingly, the immunoprecipitated CDF2-YFP complex (S8 Fig) contains pri-miR156a, pri-miR172b, pri-miR319b, and pri-miR167b, which have altered levels in the cdf2 mutant, but not pri-miR164a, which showed no change in cdf2 mutant (Fig 4B, S1 Table). In contrast, DCL1 was able to bind to all 5 pri-miRNAs examined (Fig 4B). The bindings of the DCL1 to pri-miRNAs increase in the pDCL1::DCL1-YFP/cdf2 and decrease in the line of pDCL1::DCL1-YFP/Col X p35S::CDF2-HA/Col. These results suggested that CDF2 binds mainly to the CDF2-regulated pri-miRNAs in vivo, possibly due to that CDF2 has more accessibility to these pri-miRNAs transcribed with the aid of the transcription complex containing CDF2, this was supported by a recent report that transcription and processing of primary microRNAs are coupled by Elongator complex [30], resulting in that CDF2 only affects the processing of a subset of pri-miRNAs.

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.


Related in: MedlinePlus