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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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Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate.A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ32P] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ32P] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18]. The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.
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pgen-1001225-g004: Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate.A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ32P] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ32P] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18]. The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.

Mentions: We previously reported that pre-rRNA cleaved at the primary cleavage site in the 5′ETS region (P site) accumulates in Atnuc-L1-1 mutant plants [18]. Here we examine accumulation of 45S pre-rRNA and processing in the 3′ETS region in Atnuc-L1-1 plants because 3′ETS cleavage is a co-transcriptional event that releases 45S pre-rRNA precursor [6]. To verify accumulation of these precursors in Atnuc-L1-1 plants we performed northern blot analysis using oligonucleotide probes p34 and p35 that specifically match 5′ETS sequences located either upstream and downstream from the P cleavage site, respectively, and oligonucleotide p41 to detects 3′ETS region (position shown in Figure 1 and Figure S4). As observed in Figure 4A, hybridization using p34, p35 and p41 primers generates stronger radioactive signals in the Atnuc-L1 mutant (lanes 1–2, 4–5 and 10–11) than in WT samples (lanes 3, 6 and 12). The larger radioactive signals detected using p41 (lanes 10–12), but also by p34 and p35 (lanes 1–6), correspond to the 45S pre-rRNA. The signals detected only by primer p35 correspond to an intermediate of 18S precursor forms (lanes 4–6). The smear detected with primer p34 might be due to exonucleolityc degradation of a 5′ETS cleave off product, while the signal indicated by an asterisk might correspond to a 5′ETS product produced by an alternative cleavage event upstream from the P site (lanes 1–3). Hybridization using primer p36 detects similar amounts of 18S rRNA in WT and Atnuc-L1 samples (lanes 7–9), Accumulation of pre-rRNA in Atnuc-L1-1 mutants was also observed by Northern blot using primers specific to ITS1, ITS2, 5.8S and 25S sequences. As for 18S we did not observe accumulation of either mature 5.8 or 25S rRNA (Figure S4).


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)

Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate.A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ32P] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ32P] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18]. The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.
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Related In: Results  -  Collection

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pgen-1001225-g004: Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate.A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ32P] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ32P] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18]. The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.
Mentions: We previously reported that pre-rRNA cleaved at the primary cleavage site in the 5′ETS region (P site) accumulates in Atnuc-L1-1 mutant plants [18]. Here we examine accumulation of 45S pre-rRNA and processing in the 3′ETS region in Atnuc-L1-1 plants because 3′ETS cleavage is a co-transcriptional event that releases 45S pre-rRNA precursor [6]. To verify accumulation of these precursors in Atnuc-L1-1 plants we performed northern blot analysis using oligonucleotide probes p34 and p35 that specifically match 5′ETS sequences located either upstream and downstream from the P cleavage site, respectively, and oligonucleotide p41 to detects 3′ETS region (position shown in Figure 1 and Figure S4). As observed in Figure 4A, hybridization using p34, p35 and p41 primers generates stronger radioactive signals in the Atnuc-L1 mutant (lanes 1–2, 4–5 and 10–11) than in WT samples (lanes 3, 6 and 12). The larger radioactive signals detected using p41 (lanes 10–12), but also by p34 and p35 (lanes 1–6), correspond to the 45S pre-rRNA. The signals detected only by primer p35 correspond to an intermediate of 18S precursor forms (lanes 4–6). The smear detected with primer p34 might be due to exonucleolityc degradation of a 5′ETS cleave off product, while the signal indicated by an asterisk might correspond to a 5′ETS product produced by an alternative cleavage event upstream from the P site (lanes 1–3). Hybridization using primer p36 detects similar amounts of 18S rRNA in WT and Atnuc-L1 samples (lanes 7–9), Accumulation of pre-rRNA in Atnuc-L1-1 mutants was also observed by Northern blot using primers specific to ITS1, ITS2, 5.8S and 25S sequences. As for 18S we did not observe accumulation of either mature 5.8 or 25S rRNA (Figure S4).

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