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Nsi1 plays a significant role in the silencing of ribosomal DNA in Saccharomyces cerevisiae.

Ha CW, Sung MK, Huh WK - Nucleic Acids Res. (2012)

Bottom Line: Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1.The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1.Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.

ABSTRACT
In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100-200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

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Related in: MedlinePlus

Ydr026c is associated with the NTS1 region of rDNA and is required for NTS1-specific rDNA silencing. (A) The structure of the tandemly repeating rDNA of S. cerevisiae is shown above, and a single 9.1-kb rDNA unit is shown expanded below. PCR amplicons used in ChIP assays are indicated below the rDNA unit. (B) Ydr026c and Fob1 are associated with the NTS1 region of rDNA. Shown is the degree of association of Ydr026c (solid line) and Fob1 (dashed line) with rDNA. The degree of association with rDNA was measured using ChIP assay. Relative fold enrichment refers to the relative ratio of PCR products amplified from immunoprecipitated DNA to products from input DNA. Values represent the average of three independent experiments, and error bars indicate the SEM. (C) Fob1 is required for the recruitment of Ydr026c to NTS1. Shown is the degree of association of Ydr026c with rDNA in the presence (solid line) or absence (dashed line) of Fob1. (D) Ydr026c is not required for the association of Fob1 with NTS1. Shown is the degree of association of Fob1 with rDNA in the presence (solid line) or absence (dashed line) of Ydr026c. (E) Ydr026c contributes to rDNA silencing at NTS1 but not at NTS2. Silencing within rDNA was assessed by monitoring the growth of cells (10-fold serial dilutions) plated on SC medium without uracil. SC medium was used as a plating control. (F) Ydr026c contributes to transcriptional silencing of the mURA3 reporter gene at NTS1 but not at NTS2. Total RNA was extracted from wild-type (WT), sir2Δ, fob1Δ and ydr026cΔ cells. Quantitative real-time reverse transcription–PCR analysis was performed to measure the transcript levels of the mURA3 gene inserted inside (RDN1-NTS1::mURA3 and RDN1-NTS2::mURA3) or outside the rDNA array (leu2::mURA3). Amplification efficiencies were validated and normalized against ACT1. Relative mURA3 transcript levels were calculated as the ratio of the normalized transcript level of the mURA3 reporter gene inside the NTS1 or NTS2 region to that outside the rDNA array. Primers used for the amplification of mURA3 were 5′-CTGTTGACATTGCGAAGAGC-3′ and 5′-TCTCCCTTGTCATCTAAACC-3′, and those for ACT1 were 5′-TGACTGACTACTTGATGAAG-3′ and 5′-TGCATTTCTTGTTCGAAGTC-3′. All reactions were carried out in triplicate and error bars indicate the SEM. Asterisks indicate P < 0.05, compared with wild-type cells (Student’s t-test).
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gks188-F2: Ydr026c is associated with the NTS1 region of rDNA and is required for NTS1-specific rDNA silencing. (A) The structure of the tandemly repeating rDNA of S. cerevisiae is shown above, and a single 9.1-kb rDNA unit is shown expanded below. PCR amplicons used in ChIP assays are indicated below the rDNA unit. (B) Ydr026c and Fob1 are associated with the NTS1 region of rDNA. Shown is the degree of association of Ydr026c (solid line) and Fob1 (dashed line) with rDNA. The degree of association with rDNA was measured using ChIP assay. Relative fold enrichment refers to the relative ratio of PCR products amplified from immunoprecipitated DNA to products from input DNA. Values represent the average of three independent experiments, and error bars indicate the SEM. (C) Fob1 is required for the recruitment of Ydr026c to NTS1. Shown is the degree of association of Ydr026c with rDNA in the presence (solid line) or absence (dashed line) of Fob1. (D) Ydr026c is not required for the association of Fob1 with NTS1. Shown is the degree of association of Fob1 with rDNA in the presence (solid line) or absence (dashed line) of Ydr026c. (E) Ydr026c contributes to rDNA silencing at NTS1 but not at NTS2. Silencing within rDNA was assessed by monitoring the growth of cells (10-fold serial dilutions) plated on SC medium without uracil. SC medium was used as a plating control. (F) Ydr026c contributes to transcriptional silencing of the mURA3 reporter gene at NTS1 but not at NTS2. Total RNA was extracted from wild-type (WT), sir2Δ, fob1Δ and ydr026cΔ cells. Quantitative real-time reverse transcription–PCR analysis was performed to measure the transcript levels of the mURA3 gene inserted inside (RDN1-NTS1::mURA3 and RDN1-NTS2::mURA3) or outside the rDNA array (leu2::mURA3). Amplification efficiencies were validated and normalized against ACT1. Relative mURA3 transcript levels were calculated as the ratio of the normalized transcript level of the mURA3 reporter gene inside the NTS1 or NTS2 region to that outside the rDNA array. Primers used for the amplification of mURA3 were 5′-CTGTTGACATTGCGAAGAGC-3′ and 5′-TCTCCCTTGTCATCTAAACC-3′, and those for ACT1 were 5′-TGACTGACTACTTGATGAAG-3′ and 5′-TGCATTTCTTGTTCGAAGTC-3′. All reactions were carried out in triplicate and error bars indicate the SEM. Asterisks indicate P < 0.05, compared with wild-type cells (Student’s t-test).

Mentions: The synthesis and processing of rRNAs and ribosome assembly occur in the nucleolus, and ribosomal DNA (rDNA) constitutes the basis of this organelle. In Saccharomyces cerevisiae, 100–200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII (1). Each repeat contains a Pol I-transcribed 35S rRNA gene and a non-transcribed spacer (NTS) that is divided by a Pol III-transcribed 5S rRNA gene into NTS1 and NTS2 (Figure 2A). Due to its highly repetitive nature, an rDNA array is an easy target for homologous recombination events, and recombination between rDNA repeats, which leads to the formation of extrachromosomal rDNA circles (ERCs) that accumulate to toxic levels in mother cells, is a primary cause of aging in S. cerevisiae (2). Cells have therefore evolved mechanisms to protect rDNA arrays, as their stability is critical for growth and survival. Under normal conditions, rDNA repeats remain relatively stable because the homologous recombination between them is negatively regulated through a mechanism referred to as rDNA silencing.


Nsi1 plays a significant role in the silencing of ribosomal DNA in Saccharomyces cerevisiae.

Ha CW, Sung MK, Huh WK - Nucleic Acids Res. (2012)

Ydr026c is associated with the NTS1 region of rDNA and is required for NTS1-specific rDNA silencing. (A) The structure of the tandemly repeating rDNA of S. cerevisiae is shown above, and a single 9.1-kb rDNA unit is shown expanded below. PCR amplicons used in ChIP assays are indicated below the rDNA unit. (B) Ydr026c and Fob1 are associated with the NTS1 region of rDNA. Shown is the degree of association of Ydr026c (solid line) and Fob1 (dashed line) with rDNA. The degree of association with rDNA was measured using ChIP assay. Relative fold enrichment refers to the relative ratio of PCR products amplified from immunoprecipitated DNA to products from input DNA. Values represent the average of three independent experiments, and error bars indicate the SEM. (C) Fob1 is required for the recruitment of Ydr026c to NTS1. Shown is the degree of association of Ydr026c with rDNA in the presence (solid line) or absence (dashed line) of Fob1. (D) Ydr026c is not required for the association of Fob1 with NTS1. Shown is the degree of association of Fob1 with rDNA in the presence (solid line) or absence (dashed line) of Ydr026c. (E) Ydr026c contributes to rDNA silencing at NTS1 but not at NTS2. Silencing within rDNA was assessed by monitoring the growth of cells (10-fold serial dilutions) plated on SC medium without uracil. SC medium was used as a plating control. (F) Ydr026c contributes to transcriptional silencing of the mURA3 reporter gene at NTS1 but not at NTS2. Total RNA was extracted from wild-type (WT), sir2Δ, fob1Δ and ydr026cΔ cells. Quantitative real-time reverse transcription–PCR analysis was performed to measure the transcript levels of the mURA3 gene inserted inside (RDN1-NTS1::mURA3 and RDN1-NTS2::mURA3) or outside the rDNA array (leu2::mURA3). Amplification efficiencies were validated and normalized against ACT1. Relative mURA3 transcript levels were calculated as the ratio of the normalized transcript level of the mURA3 reporter gene inside the NTS1 or NTS2 region to that outside the rDNA array. Primers used for the amplification of mURA3 were 5′-CTGTTGACATTGCGAAGAGC-3′ and 5′-TCTCCCTTGTCATCTAAACC-3′, and those for ACT1 were 5′-TGACTGACTACTTGATGAAG-3′ and 5′-TGCATTTCTTGTTCGAAGTC-3′. All reactions were carried out in triplicate and error bars indicate the SEM. Asterisks indicate P < 0.05, compared with wild-type cells (Student’s t-test).
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Related In: Results  -  Collection

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Show All Figures
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gks188-F2: Ydr026c is associated with the NTS1 region of rDNA and is required for NTS1-specific rDNA silencing. (A) The structure of the tandemly repeating rDNA of S. cerevisiae is shown above, and a single 9.1-kb rDNA unit is shown expanded below. PCR amplicons used in ChIP assays are indicated below the rDNA unit. (B) Ydr026c and Fob1 are associated with the NTS1 region of rDNA. Shown is the degree of association of Ydr026c (solid line) and Fob1 (dashed line) with rDNA. The degree of association with rDNA was measured using ChIP assay. Relative fold enrichment refers to the relative ratio of PCR products amplified from immunoprecipitated DNA to products from input DNA. Values represent the average of three independent experiments, and error bars indicate the SEM. (C) Fob1 is required for the recruitment of Ydr026c to NTS1. Shown is the degree of association of Ydr026c with rDNA in the presence (solid line) or absence (dashed line) of Fob1. (D) Ydr026c is not required for the association of Fob1 with NTS1. Shown is the degree of association of Fob1 with rDNA in the presence (solid line) or absence (dashed line) of Ydr026c. (E) Ydr026c contributes to rDNA silencing at NTS1 but not at NTS2. Silencing within rDNA was assessed by monitoring the growth of cells (10-fold serial dilutions) plated on SC medium without uracil. SC medium was used as a plating control. (F) Ydr026c contributes to transcriptional silencing of the mURA3 reporter gene at NTS1 but not at NTS2. Total RNA was extracted from wild-type (WT), sir2Δ, fob1Δ and ydr026cΔ cells. Quantitative real-time reverse transcription–PCR analysis was performed to measure the transcript levels of the mURA3 gene inserted inside (RDN1-NTS1::mURA3 and RDN1-NTS2::mURA3) or outside the rDNA array (leu2::mURA3). Amplification efficiencies were validated and normalized against ACT1. Relative mURA3 transcript levels were calculated as the ratio of the normalized transcript level of the mURA3 reporter gene inside the NTS1 or NTS2 region to that outside the rDNA array. Primers used for the amplification of mURA3 were 5′-CTGTTGACATTGCGAAGAGC-3′ and 5′-TCTCCCTTGTCATCTAAACC-3′, and those for ACT1 were 5′-TGACTGACTACTTGATGAAG-3′ and 5′-TGCATTTCTTGTTCGAAGTC-3′. All reactions were carried out in triplicate and error bars indicate the SEM. Asterisks indicate P < 0.05, compared with wild-type cells (Student’s t-test).
Mentions: The synthesis and processing of rRNAs and ribosome assembly occur in the nucleolus, and ribosomal DNA (rDNA) constitutes the basis of this organelle. In Saccharomyces cerevisiae, 100–200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII (1). Each repeat contains a Pol I-transcribed 35S rRNA gene and a non-transcribed spacer (NTS) that is divided by a Pol III-transcribed 5S rRNA gene into NTS1 and NTS2 (Figure 2A). Due to its highly repetitive nature, an rDNA array is an easy target for homologous recombination events, and recombination between rDNA repeats, which leads to the formation of extrachromosomal rDNA circles (ERCs) that accumulate to toxic levels in mother cells, is a primary cause of aging in S. cerevisiae (2). Cells have therefore evolved mechanisms to protect rDNA arrays, as their stability is critical for growth and survival. Under normal conditions, rDNA repeats remain relatively stable because the homologous recombination between them is negatively regulated through a mechanism referred to as rDNA silencing.

Bottom Line: Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1.The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1.Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.

ABSTRACT
In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100-200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

Show MeSH
Related in: MedlinePlus