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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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rRNA gene variants are differentially expressed in A. thaliana plants.A) One Step RT-PCR analysis was using primers p3/p4 to detect 3′ETS pre-rRNA sequence variants in exponentially (lane 1) or stationary growing (lane 2) cells; in roots (lane 3), flower (lane 4), leaves from Atnuc-L1-2 mutant (lane 5) or WT (lane 6) plants or 2 days germinating seeds (lane 7). RNA from roots and leaves were prepared from 15 days old WT and Atnuc-L1 plant grown in MS medium as described previously [18]. Panel eIF1α; amplification of eIF1α mRNA was performed to control PCR reaction and total RNA in different samples. B) NOR condensation is affected in 2 day germinating seeds. Left, chromatin counterstained with DAPI, Middle FISH using 25S rRNA probe and Right, superposition of FISH and DAPI images.
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pgen-1001225-g003: rRNA gene variants are differentially expressed in A. thaliana plants.A) One Step RT-PCR analysis was using primers p3/p4 to detect 3′ETS pre-rRNA sequence variants in exponentially (lane 1) or stationary growing (lane 2) cells; in roots (lane 3), flower (lane 4), leaves from Atnuc-L1-2 mutant (lane 5) or WT (lane 6) plants or 2 days germinating seeds (lane 7). RNA from roots and leaves were prepared from 15 days old WT and Atnuc-L1 plant grown in MS medium as described previously [18]. Panel eIF1α; amplification of eIF1α mRNA was performed to control PCR reaction and total RNA in different samples. B) NOR condensation is affected in 2 day germinating seeds. Left, chromatin counterstained with DAPI, Middle FISH using 25S rRNA probe and Right, superposition of FISH and DAPI images.

Mentions: As observed in Figure 3A, the 3′ETS rRNA VAR2 and VAR3 are detected in roots, flowers, leaves and germinating seeds (lanes 3, 4 and 6, 7). rRNA VAR1 is detected essentially in seeds imbibed for 48 hrs (lane 7), though a very weak band is amplified in roots and flowers (lanes 3 and 4). In exponential or stationary cell culture (lanes 1 and 2), the rRNA VAR3 corresponds to the major amplification band. As shown previously, VAR1 is also detected in Atnuc-L1 mutant plants, used here as a control (lane 5). PCR reactions without reverse transcriptase did not reveal amplification products in any of the samples tested (data not shown). Amplification of eIF1α was performed to control RNA amounts in each sample (panel eIF1α).


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)

rRNA gene variants are differentially expressed in A. thaliana plants.A) One Step RT-PCR analysis was using primers p3/p4 to detect 3′ETS pre-rRNA sequence variants in exponentially (lane 1) or stationary growing (lane 2) cells; in roots (lane 3), flower (lane 4), leaves from Atnuc-L1-2 mutant (lane 5) or WT (lane 6) plants or 2 days germinating seeds (lane 7). RNA from roots and leaves were prepared from 15 days old WT and Atnuc-L1 plant grown in MS medium as described previously [18]. Panel eIF1α; amplification of eIF1α mRNA was performed to control PCR reaction and total RNA in different samples. B) NOR condensation is affected in 2 day germinating seeds. Left, chromatin counterstained with DAPI, Middle FISH using 25S rRNA probe and Right, superposition of FISH and DAPI images.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991258&req=5

pgen-1001225-g003: rRNA gene variants are differentially expressed in A. thaliana plants.A) One Step RT-PCR analysis was using primers p3/p4 to detect 3′ETS pre-rRNA sequence variants in exponentially (lane 1) or stationary growing (lane 2) cells; in roots (lane 3), flower (lane 4), leaves from Atnuc-L1-2 mutant (lane 5) or WT (lane 6) plants or 2 days germinating seeds (lane 7). RNA from roots and leaves were prepared from 15 days old WT and Atnuc-L1 plant grown in MS medium as described previously [18]. Panel eIF1α; amplification of eIF1α mRNA was performed to control PCR reaction and total RNA in different samples. B) NOR condensation is affected in 2 day germinating seeds. Left, chromatin counterstained with DAPI, Middle FISH using 25S rRNA probe and Right, superposition of FISH and DAPI images.
Mentions: As observed in Figure 3A, the 3′ETS rRNA VAR2 and VAR3 are detected in roots, flowers, leaves and germinating seeds (lanes 3, 4 and 6, 7). rRNA VAR1 is detected essentially in seeds imbibed for 48 hrs (lane 7), though a very weak band is amplified in roots and flowers (lanes 3 and 4). In exponential or stationary cell culture (lanes 1 and 2), the rRNA VAR3 corresponds to the major amplification band. As shown previously, VAR1 is also detected in Atnuc-L1 mutant plants, used here as a control (lane 5). PCR reactions without reverse transcriptase did not reveal amplification products in any of the samples tested (data not shown). Amplification of eIF1α was performed to control RNA amounts in each sample (panel eIF1α).

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
Related in: MedlinePlus