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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 acts as a transcription factor for some miRNA genes.(A) The relative levels of pri-miRNAs in Col, cdf2, p35S::CDF2/Col, p35S::DCL1/Col lines examined by real-time PCR. The relative fold changes were normalized to ACTIN. Data are given as means ± SD (n = 3). (B) ChIP-PCR analysis of five promoter fragments of miRNA genes in wild-type and pCDF2:CDF2-YFP/Col seedlings. ChIP assays were performed using the 22-day-old Col-0 and pCDF2::CDF2-YFP/Col seedlings expressing the CDF2-YFP fusion protein. DNA was amplified using primers specific to 6 miRNA promoter regions. (C) CHIP followed by real time PCR of 6 promoter fragments of miRNA genes in Col and pCDF2::CDF2-YFP/Col seedlings. Relative enrichment of fragments was calculated with HA antibodies as the control. Data are given as means ± SD (n = 3). (D) and (E) pMIR172a::GUS in Col, cdf2 and p35S::DCL1/Col in seedlings (D) and flowers (E), respectively. Thirty plants containing GUS were analyzed for each of genotypes. (F) The transcript levels of GUS driven by miR172b promoter in Col, cdf2 and p35S::DCL1/Col. GUS transcript levels were determined by qRT-PCR. Data are given as means ± SD (n = 3).
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pgen.1005598.g003: CDF2 acts as a transcription factor for some miRNA genes.(A) The relative levels of pri-miRNAs in Col, cdf2, p35S::CDF2/Col, p35S::DCL1/Col lines examined by real-time PCR. The relative fold changes were normalized to ACTIN. Data are given as means ± SD (n = 3). (B) ChIP-PCR analysis of five promoter fragments of miRNA genes in wild-type and pCDF2:CDF2-YFP/Col seedlings. ChIP assays were performed using the 22-day-old Col-0 and pCDF2::CDF2-YFP/Col seedlings expressing the CDF2-YFP fusion protein. DNA was amplified using primers specific to 6 miRNA promoter regions. (C) CHIP followed by real time PCR of 6 promoter fragments of miRNA genes in Col and pCDF2::CDF2-YFP/Col seedlings. Relative enrichment of fragments was calculated with HA antibodies as the control. Data are given as means ± SD (n = 3). (D) and (E) pMIR172a::GUS in Col, cdf2 and p35S::DCL1/Col in seedlings (D) and flowers (E), respectively. Thirty plants containing GUS were analyzed for each of genotypes. (F) The transcript levels of GUS driven by miR172b promoter in Col, cdf2 and p35S::DCL1/Col. GUS transcript levels were determined by qRT-PCR. Data are given as means ± SD (n = 3).

Mentions: To address the molecular mechanism of the effect of CDF2 on miRNA abundance, we first examined the expressional levels of DCL1 and HYL1, the two main components involved in miRNA biogenesis, in cdf2 mutant and CDF2 overexpression lines (S6 Fig), the result shows that the expression levels of the genes are similar to those in Col. We then analyzed the levels of 19 pri-miRNAs, for which their mature miRNAs show at least 1.5-fold differences (S1 Table), by quantitative real-time PCR (qRT-PCR) in seedlings of cdf2 mutant and CDF2-overexpressing (p35S::CDF2/Col) lines. As shown in Fig 3A, the relative levels of pri-miRNAs between cdf2 and p35S::CDF2/Col are predominantly opposite for all 19 pri-miRNAs. Some of pri-miRNAs (pri-mi172a and pri-miR394a) are up-regulated, while others down-regulated in cdf2 mutant (Fig 3A), indicating that CDF2 acts as both a transcriptional activator and repressor, which is similar to other Dof proteins, e.g., maize Dof2 exhibits either activation or repression activities on different promoters [28]; the barley Dof factor (prolamin-box binding factor) activates the transcription of B-hordein, but represses the transcription of cathepsin B-like protease [29]. The distinct roles of CDF2 in miRNA transcription may be defined by the components forming different transcriptional machinery to be recruited to different promoters.


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 acts as a transcription factor for some miRNA genes.(A) The relative levels of pri-miRNAs in Col, cdf2, p35S::CDF2/Col, p35S::DCL1/Col lines examined by real-time PCR. The relative fold changes were normalized to ACTIN. Data are given as means ± SD (n = 3). (B) ChIP-PCR analysis of five promoter fragments of miRNA genes in wild-type and pCDF2:CDF2-YFP/Col seedlings. ChIP assays were performed using the 22-day-old Col-0 and pCDF2::CDF2-YFP/Col seedlings expressing the CDF2-YFP fusion protein. DNA was amplified using primers specific to 6 miRNA promoter regions. (C) CHIP followed by real time PCR of 6 promoter fragments of miRNA genes in Col and pCDF2::CDF2-YFP/Col seedlings. Relative enrichment of fragments was calculated with HA antibodies as the control. Data are given as means ± SD (n = 3). (D) and (E) pMIR172a::GUS in Col, cdf2 and p35S::DCL1/Col in seedlings (D) and flowers (E), respectively. Thirty plants containing GUS were analyzed for each of genotypes. (F) The transcript levels of GUS driven by miR172b promoter in Col, cdf2 and p35S::DCL1/Col. GUS transcript levels were determined by qRT-PCR. Data are given as means ± SD (n = 3).
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Related In: Results  -  Collection

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pgen.1005598.g003: CDF2 acts as a transcription factor for some miRNA genes.(A) The relative levels of pri-miRNAs in Col, cdf2, p35S::CDF2/Col, p35S::DCL1/Col lines examined by real-time PCR. The relative fold changes were normalized to ACTIN. Data are given as means ± SD (n = 3). (B) ChIP-PCR analysis of five promoter fragments of miRNA genes in wild-type and pCDF2:CDF2-YFP/Col seedlings. ChIP assays were performed using the 22-day-old Col-0 and pCDF2::CDF2-YFP/Col seedlings expressing the CDF2-YFP fusion protein. DNA was amplified using primers specific to 6 miRNA promoter regions. (C) CHIP followed by real time PCR of 6 promoter fragments of miRNA genes in Col and pCDF2::CDF2-YFP/Col seedlings. Relative enrichment of fragments was calculated with HA antibodies as the control. Data are given as means ± SD (n = 3). (D) and (E) pMIR172a::GUS in Col, cdf2 and p35S::DCL1/Col in seedlings (D) and flowers (E), respectively. Thirty plants containing GUS were analyzed for each of genotypes. (F) The transcript levels of GUS driven by miR172b promoter in Col, cdf2 and p35S::DCL1/Col. GUS transcript levels were determined by qRT-PCR. Data are given as means ± SD (n = 3).
Mentions: To address the molecular mechanism of the effect of CDF2 on miRNA abundance, we first examined the expressional levels of DCL1 and HYL1, the two main components involved in miRNA biogenesis, in cdf2 mutant and CDF2 overexpression lines (S6 Fig), the result shows that the expression levels of the genes are similar to those in Col. We then analyzed the levels of 19 pri-miRNAs, for which their mature miRNAs show at least 1.5-fold differences (S1 Table), by quantitative real-time PCR (qRT-PCR) in seedlings of cdf2 mutant and CDF2-overexpressing (p35S::CDF2/Col) lines. As shown in Fig 3A, the relative levels of pri-miRNAs between cdf2 and p35S::CDF2/Col are predominantly opposite for all 19 pri-miRNAs. Some of pri-miRNAs (pri-mi172a and pri-miR394a) are up-regulated, while others down-regulated in cdf2 mutant (Fig 3A), indicating that CDF2 acts as both a transcriptional activator and repressor, which is similar to other Dof proteins, e.g., maize Dof2 exhibits either activation or repression activities on different promoters [28]; the barley Dof factor (prolamin-box binding factor) activates the transcription of B-hordein, but represses the transcription of cathepsin B-like protease [29]. The distinct roles of CDF2 in miRNA transcription may be defined by the components forming different transcriptional machinery to be recruited to different promoters.

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