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Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

Pontvianne F, Abou-Ellail M, Douet J, Comella P, Matia I, Chandrasekhara C, Debures A, Blevins T, Cooke R, Medina FJ, Tourmente S, Pikaard CS, Sáez-Vásquez J - PLoS Genet. (2010)

Bottom Line: We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately.Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes.Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: UMR 5096 CNRS-IRD-University de Perpignan, Perpignan, France.

ABSTRACT
In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

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AtNUC-L1 gene disruption induces accumulation of siRNA 45S and rRNA gene hypomethylation on the 5′ETS.A) Total RNA from WT (lane 2) and Atnuc-L1 (lanes 1 and 3) plants were fractionated on PAGE and hybridized with [γ32P] 5′-end labeled primers to detect siRNA (45S, AtSN1) and miR159. Hybridization to detect snRNA U6 was performed to control RNA loading in each sample. B) Bisulfite sequencing analysis. The bar graphs show the representation (%) of methylated sites in the in 5′ETS rRNA gene sequences (from -+1 to +243) from WT, Atnuc-L1 mutant and F1 WT x Atnuc-L1-2 backcrossed plants in a CG (upper panel), CHG (middle panel) and CHH (lower panel) context. C) The schema shows the position of primers p44/p45 (located at −315 and at +243) and oligonucleotide 45S siRNA (from −33 to +6) used for sequencing bisulfite treated samples and for northern blot respectively. Conserved A123B motif in the 5′ETS is shown.
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pgen-1001225-g007: AtNUC-L1 gene disruption induces accumulation of siRNA 45S and rRNA gene hypomethylation on the 5′ETS.A) Total RNA from WT (lane 2) and Atnuc-L1 (lanes 1 and 3) plants were fractionated on PAGE and hybridized with [γ32P] 5′-end labeled primers to detect siRNA (45S, AtSN1) and miR159. Hybridization to detect snRNA U6 was performed to control RNA loading in each sample. B) Bisulfite sequencing analysis. The bar graphs show the representation (%) of methylated sites in the in 5′ETS rRNA gene sequences (from -+1 to +243) from WT, Atnuc-L1 mutant and F1 WT x Atnuc-L1-2 backcrossed plants in a CG (upper panel), CHG (middle panel) and CHH (lower panel) context. C) The schema shows the position of primers p44/p45 (located at −315 and at +243) and oligonucleotide 45S siRNA (from −33 to +6) used for sequencing bisulfite treated samples and for northern blot respectively. Conserved A123B motif in the 5′ETS is shown.

Mentions: To determine if specific siRNAs to 45S rRNAs sequences accumulate in Atnuc-L1 plants, we performed northern blot experiments using specific probes for promoter 45S rRNA sequences [39]. As shown in Figure 7A, the 45S siRNA probe hybridizes to a small RNA that accumulates ∼1.8 and ∼1.7 fold in Atnuc-L1-1 and Atnuc-L1-2 mutant plants respectively (lanes 3 and 1) compared with WT samples (lane 2). Higher levels of 45S siRNA were also observed in Northern blot experiments using total RNA extracted from WT and Atnuc-L1 flower buds (data not shown). Hybridization with primers corresponding to mir159 and AtSN1 siRNA did not detect major differences between WT and Atnuc-L1 samples (Panel miR159 and AtSN1). Hybridization with primers specific to small nuclear RNA U6 was used as an RNA loading control (panel snRNA U6).


Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

Pontvianne F, Abou-Ellail M, Douet J, Comella P, Matia I, Chandrasekhara C, Debures A, Blevins T, Cooke R, Medina FJ, Tourmente S, Pikaard CS, Sáez-Vásquez J - PLoS Genet. (2010)

AtNUC-L1 gene disruption induces accumulation of siRNA 45S and rRNA gene hypomethylation on the 5′ETS.A) Total RNA from WT (lane 2) and Atnuc-L1 (lanes 1 and 3) plants were fractionated on PAGE and hybridized with [γ32P] 5′-end labeled primers to detect siRNA (45S, AtSN1) and miR159. Hybridization to detect snRNA U6 was performed to control RNA loading in each sample. B) Bisulfite sequencing analysis. The bar graphs show the representation (%) of methylated sites in the in 5′ETS rRNA gene sequences (from -+1 to +243) from WT, Atnuc-L1 mutant and F1 WT x Atnuc-L1-2 backcrossed plants in a CG (upper panel), CHG (middle panel) and CHH (lower panel) context. C) The schema shows the position of primers p44/p45 (located at −315 and at +243) and oligonucleotide 45S siRNA (from −33 to +6) used for sequencing bisulfite treated samples and for northern blot respectively. Conserved A123B motif in the 5′ETS is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991258&req=5

pgen-1001225-g007: AtNUC-L1 gene disruption induces accumulation of siRNA 45S and rRNA gene hypomethylation on the 5′ETS.A) Total RNA from WT (lane 2) and Atnuc-L1 (lanes 1 and 3) plants were fractionated on PAGE and hybridized with [γ32P] 5′-end labeled primers to detect siRNA (45S, AtSN1) and miR159. Hybridization to detect snRNA U6 was performed to control RNA loading in each sample. B) Bisulfite sequencing analysis. The bar graphs show the representation (%) of methylated sites in the in 5′ETS rRNA gene sequences (from -+1 to +243) from WT, Atnuc-L1 mutant and F1 WT x Atnuc-L1-2 backcrossed plants in a CG (upper panel), CHG (middle panel) and CHH (lower panel) context. C) The schema shows the position of primers p44/p45 (located at −315 and at +243) and oligonucleotide 45S siRNA (from −33 to +6) used for sequencing bisulfite treated samples and for northern blot respectively. Conserved A123B motif in the 5′ETS is shown.
Mentions: To determine if specific siRNAs to 45S rRNAs sequences accumulate in Atnuc-L1 plants, we performed northern blot experiments using specific probes for promoter 45S rRNA sequences [39]. As shown in Figure 7A, the 45S siRNA probe hybridizes to a small RNA that accumulates ∼1.8 and ∼1.7 fold in Atnuc-L1-1 and Atnuc-L1-2 mutant plants respectively (lanes 3 and 1) compared with WT samples (lane 2). Higher levels of 45S siRNA were also observed in Northern blot experiments using total RNA extracted from WT and Atnuc-L1 flower buds (data not shown). Hybridization with primers corresponding to mir159 and AtSN1 siRNA did not detect major differences between WT and Atnuc-L1 samples (Panel miR159 and AtSN1). Hybridization with primers specific to small nuclear RNA U6 was used as an RNA loading control (panel snRNA U6).

Bottom Line: We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately.Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes.Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: UMR 5096 CNRS-IRD-University de Perpignan, Perpignan, France.

ABSTRACT
In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

Show MeSH