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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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Long 5S rDNA-derived transcripts are subject to RNA silencing.A) Map of Arabidopsis small RNAs matching the 5S rDNA unit repeat, based on analysis of leaf datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 5′-end positions are indicated on the x-axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom shows a 5S rRNA gene (thick black arrow), the surrounding intergenic spacers (IGS, gray), and regions probed by RNA blot hybridization (red lines). RT-PCR products obtained with F and R primers are indicated (black lines). B) Searching small RNA datasets for the siR1003 core sequence (yellow box) yielded a family of 21 to 24-nt siRNAs, which all match 5S LT1 transcripts identified by Vaillant et al. (2006) [52]. C) Analysis of 5S LT1 transcript accumulation: RNA samples from inflorescences of wild type (WT), ddm1 and siRNA biogenesis mutants were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (panel A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). D) Blot analysis of small RNA isolated from material described in panel C. The membrane was sequentially hybridized with a DNA oligonucleotide (oligo) that detects siR1003, an RNA probe for the 3′-flanking IGS region (panel A, diagram), a DNA oligo that detects miR160, and DNA oligos that detect U6 snRNA. Migration of 21-nt and 24-nt RNA oligo size standards is indicated at left.
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pone-0005932-g001: Long 5S rDNA-derived transcripts are subject to RNA silencing.A) Map of Arabidopsis small RNAs matching the 5S rDNA unit repeat, based on analysis of leaf datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 5′-end positions are indicated on the x-axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom shows a 5S rRNA gene (thick black arrow), the surrounding intergenic spacers (IGS, gray), and regions probed by RNA blot hybridization (red lines). RT-PCR products obtained with F and R primers are indicated (black lines). B) Searching small RNA datasets for the siR1003 core sequence (yellow box) yielded a family of 21 to 24-nt siRNAs, which all match 5S LT1 transcripts identified by Vaillant et al. (2006) [52]. C) Analysis of 5S LT1 transcript accumulation: RNA samples from inflorescences of wild type (WT), ddm1 and siRNA biogenesis mutants were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (panel A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). D) Blot analysis of small RNA isolated from material described in panel C. The membrane was sequentially hybridized with a DNA oligonucleotide (oligo) that detects siR1003, an RNA probe for the 3′-flanking IGS region (panel A, diagram), a DNA oligo that detects miR160, and DNA oligos that detect U6 snRNA. Migration of 21-nt and 24-nt RNA oligo size standards is indicated at left.

Mentions: Analysis of the origin of siRNAs is complicated by natural variation among the ∼1000 5S rDNA repeats. To resolve this problem, we generated a gapped alignment of 283 individual 5S rDNA repeats derived from an A. thaliana YAC library [58]. Next, 5′-end positions of individually matched small RNAs were aligned with a composite 530-bp repeat. Figure 1A summarizes our analysis of small RNA datasets from leaves obtained by Rajagopalan et al. (2006) and Kasschau et al. (2007) [57], [59]. Interestingly, 60% of the 3662 matched sequencing reads represented the intergenic spacer (IGS). A cluster of small RNAs (Figure 1A, **) in the IGS matched both strands (44% and 56%, respectively) and included siR1003 (Figure 1B). This IGS cluster was also apparent in the maps we generated for other Arabidopsis tissues (Figure S1, seedlings and inflorescences). The remaining sequences that matched 5S rDNA, 40%, correspond to the 120-bp 5S rRNA genes: these reads were disproportionately 20 to 22-nt long and corresponded predominantly to the 5S rRNA-encoding strand (92% of 1481 genic reads). Analysis of inflorescence data yielded a spike of reads with 5′-ends corresponding to the 5S rRNA 5′-terminus (Figure S1). Together with the abundant reads with 3′-ends aligned to the 5S rRNA 3′-terminus (Figure 1A and Figure S1), this strongly suggests that most genic small RNAs are products of 5S rRNA degradation.


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

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

Long 5S rDNA-derived transcripts are subject to RNA silencing.A) Map of Arabidopsis small RNAs matching the 5S rDNA unit repeat, based on analysis of leaf datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 5′-end positions are indicated on the x-axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom shows a 5S rRNA gene (thick black arrow), the surrounding intergenic spacers (IGS, gray), and regions probed by RNA blot hybridization (red lines). RT-PCR products obtained with F and R primers are indicated (black lines). B) Searching small RNA datasets for the siR1003 core sequence (yellow box) yielded a family of 21 to 24-nt siRNAs, which all match 5S LT1 transcripts identified by Vaillant et al. (2006) [52]. C) Analysis of 5S LT1 transcript accumulation: RNA samples from inflorescences of wild type (WT), ddm1 and siRNA biogenesis mutants were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (panel A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). D) Blot analysis of small RNA isolated from material described in panel C. The membrane was sequentially hybridized with a DNA oligonucleotide (oligo) that detects siR1003, an RNA probe for the 3′-flanking IGS region (panel A, diagram), a DNA oligo that detects miR160, and DNA oligos that detect U6 snRNA. Migration of 21-nt and 24-nt RNA oligo size standards is indicated at left.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005932-g001: Long 5S rDNA-derived transcripts are subject to RNA silencing.A) Map of Arabidopsis small RNAs matching the 5S rDNA unit repeat, based on analysis of leaf datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 5′-end positions are indicated on the x-axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom shows a 5S rRNA gene (thick black arrow), the surrounding intergenic spacers (IGS, gray), and regions probed by RNA blot hybridization (red lines). RT-PCR products obtained with F and R primers are indicated (black lines). B) Searching small RNA datasets for the siR1003 core sequence (yellow box) yielded a family of 21 to 24-nt siRNAs, which all match 5S LT1 transcripts identified by Vaillant et al. (2006) [52]. C) Analysis of 5S LT1 transcript accumulation: RNA samples from inflorescences of wild type (WT), ddm1 and siRNA biogenesis mutants were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (panel A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). D) Blot analysis of small RNA isolated from material described in panel C. The membrane was sequentially hybridized with a DNA oligonucleotide (oligo) that detects siR1003, an RNA probe for the 3′-flanking IGS region (panel A, diagram), a DNA oligo that detects miR160, and DNA oligos that detect U6 snRNA. Migration of 21-nt and 24-nt RNA oligo size standards is indicated at left.
Mentions: Analysis of the origin of siRNAs is complicated by natural variation among the ∼1000 5S rDNA repeats. To resolve this problem, we generated a gapped alignment of 283 individual 5S rDNA repeats derived from an A. thaliana YAC library [58]. Next, 5′-end positions of individually matched small RNAs were aligned with a composite 530-bp repeat. Figure 1A summarizes our analysis of small RNA datasets from leaves obtained by Rajagopalan et al. (2006) and Kasschau et al. (2007) [57], [59]. Interestingly, 60% of the 3662 matched sequencing reads represented the intergenic spacer (IGS). A cluster of small RNAs (Figure 1A, **) in the IGS matched both strands (44% and 56%, respectively) and included siR1003 (Figure 1B). This IGS cluster was also apparent in the maps we generated for other Arabidopsis tissues (Figure S1, seedlings and inflorescences). The remaining sequences that matched 5S rDNA, 40%, correspond to the 120-bp 5S rRNA genes: these reads were disproportionately 20 to 22-nt long and corresponded predominantly to the 5S rRNA-encoding strand (92% of 1481 genic reads). Analysis of inflorescence data yielded a spike of reads with 5′-ends corresponding to the 5S rRNA 5′-terminus (Figure S1). Together with the abundant reads with 3′-ends aligned to the 5S rRNA 3′-terminus (Figure 1A and Figure S1), this strongly suggests that most genic small RNAs are products of 5S rRNA degradation.

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