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Heterochromatic siRNAs and DDM1 independently silence aberrant 5S rDNA transcripts in Arabidopsis.

Blevins T, Pontes O, Pikaard CS, Meins F - PLoS ONE (2009)

Bottom Line: Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent.DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats.We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT
5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

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DDM1 limits IGS siRNA accumulation and asymmetric methylation.A) Detection of IGS siRNAs (siR1003) in dicer-like (dcl) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4; double mutants dcl2 dcl3 (dcl2/3), dcl2 dcl4 (dcl2/4), dcl3 dcl4 (dcl3/4); and the triple mutant dcl2 dcl3 dcl4 (dcl2/3/4). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region (Figure 1A, diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1. Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1. Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1, rdr2, two alleles of dcl3, met1 and ddm1. The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.
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pone-0005932-g002: DDM1 limits IGS siRNA accumulation and asymmetric methylation.A) Detection of IGS siRNAs (siR1003) in dicer-like (dcl) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4; double mutants dcl2 dcl3 (dcl2/3), dcl2 dcl4 (dcl2/4), dcl3 dcl4 (dcl3/4); and the triple mutant dcl2 dcl3 dcl4 (dcl2/3/4). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region (Figure 1A, diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1. Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1. Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1, rdr2, two alleles of dcl3, met1 and ddm1. The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.

Mentions: To infer processing functions for individual DCLs, we analyzed siR1003 accumulation in double mutant combinations of three DCLs (DCL2, DCL3 and DCL4). We detected ∼22-nt siRNAs in dcl3, but this size-class was absent in the double mutant dcl2/3. Moreover, ∼21-nt species detected in dcl2/3 were absent in the triple mutant dcl2/3/4 (Figure 2A). These hierarchical DCL dependencies, previously recognized for other repeat-derived and viral siRNAs [61]–[64], suggest that 21-nt and 22-nt IGS siRNAs are products of DCL4 and DCL2, respectively. Essentially the same results were obtained using probes for the reverse complement of siR1003 (data not shown). Together, our data suggest that dsRNA derived from aberrant 5S rDNA transcripts can be processed by multiple DCLs when DCL3 is mutated, generating IGS siRNAs that contribute to silencing of 5S rDNA.


Heterochromatic siRNAs and DDM1 independently silence aberrant 5S rDNA transcripts in Arabidopsis.

Blevins T, Pontes O, Pikaard CS, Meins F - PLoS ONE (2009)

DDM1 limits IGS siRNA accumulation and asymmetric methylation.A) Detection of IGS siRNAs (siR1003) in dicer-like (dcl) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4; double mutants dcl2 dcl3 (dcl2/3), dcl2 dcl4 (dcl2/4), dcl3 dcl4 (dcl3/4); and the triple mutant dcl2 dcl3 dcl4 (dcl2/3/4). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region (Figure 1A, diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1. Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1. Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1, rdr2, two alleles of dcl3, met1 and ddm1. The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691480&req=5

pone-0005932-g002: DDM1 limits IGS siRNA accumulation and asymmetric methylation.A) Detection of IGS siRNAs (siR1003) in dicer-like (dcl) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4; double mutants dcl2 dcl3 (dcl2/3), dcl2 dcl4 (dcl2/4), dcl3 dcl4 (dcl3/4); and the triple mutant dcl2 dcl3 dcl4 (dcl2/3/4). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region (Figure 1A, diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1. Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1. Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1, rdr2, two alleles of dcl3, met1 and ddm1. The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.
Mentions: To infer processing functions for individual DCLs, we analyzed siR1003 accumulation in double mutant combinations of three DCLs (DCL2, DCL3 and DCL4). We detected ∼22-nt siRNAs in dcl3, but this size-class was absent in the double mutant dcl2/3. Moreover, ∼21-nt species detected in dcl2/3 were absent in the triple mutant dcl2/3/4 (Figure 2A). These hierarchical DCL dependencies, previously recognized for other repeat-derived and viral siRNAs [61]–[64], suggest that 21-nt and 22-nt IGS siRNAs are products of DCL4 and DCL2, respectively. Essentially the same results were obtained using probes for the reverse complement of siR1003 (data not shown). Together, our data suggest that dsRNA derived from aberrant 5S rDNA transcripts can be processed by multiple DCLs when DCL3 is mutated, generating IGS siRNAs that contribute to silencing of 5S rDNA.

Bottom Line: Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent.DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats.We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT
5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

Show MeSH
Related in: MedlinePlus