Limits...
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.

Show MeSH
Accumulation of RNA polymerase I and rRNA transcripts from the IGS in Atnuc-L1 plants.A) Chromatin samples prepared from WT (grey bars) and Atnuc-L1-1 mutant (black bars) plants were immunoprecipitated with antibodies against the largest RNA pol I subunit. ChIP samples were amplified with specific primers to GP (p10/p11), 5′ETS (p12/p13), 25S (p14/p15), 3′ETS (p16/p17) and IGS2 (p18/p19) rRNA sequences. Amplification of the ACT2 gene was performed to control ChiP and qRT-PCR reactions. B) Primer extension was performed using total RNA extracted from WT (lane 1) and AtnucL-1 (lane 2) plants and primer p6 for mapping transcription initiation site (TIS) from the gene promoter (GP). Primer extension using primer 3AtU3 that accurately maps the 5′ end of U3snoRNA was used to control similar amounts of total RNA in each reaction. Lanes 3–6 show DNA sequencing reactions used to map accurately the transcription initiation site (panel TIS) or verify the expected size for U3 snoRNA amplification. C) RT-PCR analysis using primers p42/p43 and p7/p8 to detect transcription from IGS(from −507 to −201 and from −223 to +243 respectively). One Step RT-PCR reaction of ACT2 transcripts was performed to control similar amount of total RNA in WT (lane 1) and Atnuc-L1 (lanes 2 and 3) samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991258&req=5

pgen-1001225-g005: Accumulation of RNA polymerase I and rRNA transcripts from the IGS in Atnuc-L1 plants.A) Chromatin samples prepared from WT (grey bars) and Atnuc-L1-1 mutant (black bars) plants were immunoprecipitated with antibodies against the largest RNA pol I subunit. ChIP samples were amplified with specific primers to GP (p10/p11), 5′ETS (p12/p13), 25S (p14/p15), 3′ETS (p16/p17) and IGS2 (p18/p19) rRNA sequences. Amplification of the ACT2 gene was performed to control ChiP and qRT-PCR reactions. B) Primer extension was performed using total RNA extracted from WT (lane 1) and AtnucL-1 (lane 2) plants and primer p6 for mapping transcription initiation site (TIS) from the gene promoter (GP). Primer extension using primer 3AtU3 that accurately maps the 5′ end of U3snoRNA was used to control similar amounts of total RNA in each reaction. Lanes 3–6 show DNA sequencing reactions used to map accurately the transcription initiation site (panel TIS) or verify the expected size for U3 snoRNA amplification. C) RT-PCR analysis using primers p42/p43 and p7/p8 to detect transcription from IGS(from −507 to −201 and from −223 to +243 respectively). One Step RT-PCR reaction of ACT2 transcripts was performed to control similar amount of total RNA in WT (lane 1) and Atnuc-L1 (lanes 2 and 3) samples.

Mentions: To compare the level of RNA Pol I associated with rRNA genes in WT and Atnuc-L1 plants we performed ChIP using antibodies against the largest RNA pol I subunit and specific primers to amplify Gene Promoter (GP, p10/p11), 5′External Transcribed Spacer (5′ETS, p12/p13), 25S rRNA (25S, p14/p15), 3′External Transcribed Spacer (3′ETS, p16/p17) and Internal Gene Spacer 2 (IGS2, p18/p19) sequences (Figure 5A). Quantitative PCR analysis revealed higher GP, 5′ETS, 25S and 3′ETS amplification signals in samples from Atnuc-L1-1 plants (black bars) compared to signals in samples from WT plants (grey bars). In contrast, when the region located just downstream from the four repeat sequences (IGS2) was analyzed, the PCR amplification signals in samples from Atnuc-L1 and WT plants were similar.


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)

Accumulation of RNA polymerase I and rRNA transcripts from the IGS in Atnuc-L1 plants.A) Chromatin samples prepared from WT (grey bars) and Atnuc-L1-1 mutant (black bars) plants were immunoprecipitated with antibodies against the largest RNA pol I subunit. ChIP samples were amplified with specific primers to GP (p10/p11), 5′ETS (p12/p13), 25S (p14/p15), 3′ETS (p16/p17) and IGS2 (p18/p19) rRNA sequences. Amplification of the ACT2 gene was performed to control ChiP and qRT-PCR reactions. B) Primer extension was performed using total RNA extracted from WT (lane 1) and AtnucL-1 (lane 2) plants and primer p6 for mapping transcription initiation site (TIS) from the gene promoter (GP). Primer extension using primer 3AtU3 that accurately maps the 5′ end of U3snoRNA was used to control similar amounts of total RNA in each reaction. Lanes 3–6 show DNA sequencing reactions used to map accurately the transcription initiation site (panel TIS) or verify the expected size for U3 snoRNA amplification. C) RT-PCR analysis using primers p42/p43 and p7/p8 to detect transcription from IGS(from −507 to −201 and from −223 to +243 respectively). One Step RT-PCR reaction of ACT2 transcripts was performed to control similar amount of total RNA in WT (lane 1) and Atnuc-L1 (lanes 2 and 3) samples.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001225-g005: Accumulation of RNA polymerase I and rRNA transcripts from the IGS in Atnuc-L1 plants.A) Chromatin samples prepared from WT (grey bars) and Atnuc-L1-1 mutant (black bars) plants were immunoprecipitated with antibodies against the largest RNA pol I subunit. ChIP samples were amplified with specific primers to GP (p10/p11), 5′ETS (p12/p13), 25S (p14/p15), 3′ETS (p16/p17) and IGS2 (p18/p19) rRNA sequences. Amplification of the ACT2 gene was performed to control ChiP and qRT-PCR reactions. B) Primer extension was performed using total RNA extracted from WT (lane 1) and AtnucL-1 (lane 2) plants and primer p6 for mapping transcription initiation site (TIS) from the gene promoter (GP). Primer extension using primer 3AtU3 that accurately maps the 5′ end of U3snoRNA was used to control similar amounts of total RNA in each reaction. Lanes 3–6 show DNA sequencing reactions used to map accurately the transcription initiation site (panel TIS) or verify the expected size for U3 snoRNA amplification. C) RT-PCR analysis using primers p42/p43 and p7/p8 to detect transcription from IGS(from −507 to −201 and from −223 to +243 respectively). One Step RT-PCR reaction of ACT2 transcripts was performed to control similar amount of total RNA in WT (lane 1) and Atnuc-L1 (lanes 2 and 3) samples.
Mentions: To compare the level of RNA Pol I associated with rRNA genes in WT and Atnuc-L1 plants we performed ChIP using antibodies against the largest RNA pol I subunit and specific primers to amplify Gene Promoter (GP, p10/p11), 5′External Transcribed Spacer (5′ETS, p12/p13), 25S rRNA (25S, p14/p15), 3′External Transcribed Spacer (3′ETS, p16/p17) and Internal Gene Spacer 2 (IGS2, p18/p19) sequences (Figure 5A). Quantitative PCR analysis revealed higher GP, 5′ETS, 25S and 3′ETS amplification signals in samples from Atnuc-L1-1 plants (black bars) compared to signals in samples from WT plants (grey bars). In contrast, when the region located just downstream from the four repeat sequences (IGS2) was analyzed, the PCR amplification signals in samples from Atnuc-L1 and WT plants were similar.

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