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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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Nsi1 physically interacts with Net1, Sir2 and Fob1, and the loss of Nsi1 decreases the interaction between Fob1 and Sir2. (A) Western blots showing that HA-Nsi1 coprecipitates with Net1-Myc (left panel), Sir2-Myc (middle panel) and Fob1-Myc (right panel) from whole cell extracts. (B) GST pull-down assay showing direct association of Nsi1 with Net1, Sir2 and Fob1. GST-Nsi1 associates with Net1, Sir2 and Fob1 from whole cell extracts (left panel). GST-Sir2 and GST-Fob1 associate with Nsi1 from whole cell extracts (right panel). (C) Western blots showing that the loss of Nsi1 decreases the amount of Sir2-Myc that coprecipitates with Fob1-TAP. Hexokinase was used as a loading control in all blots. The level of Sir2-Myc coprecipitated with Fob1-TAP in the presence or absence of Nsi1 was quantified and is shown to the right of the blots. Values represent the average of three independent experiments, and error bars indicate the SEM. Asterisk indicates P < 0.05, compared with wild-type cells (Student’s t-test).
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gks188-F3: Nsi1 physically interacts with Net1, Sir2 and Fob1, and the loss of Nsi1 decreases the interaction between Fob1 and Sir2. (A) Western blots showing that HA-Nsi1 coprecipitates with Net1-Myc (left panel), Sir2-Myc (middle panel) and Fob1-Myc (right panel) from whole cell extracts. (B) GST pull-down assay showing direct association of Nsi1 with Net1, Sir2 and Fob1. GST-Nsi1 associates with Net1, Sir2 and Fob1 from whole cell extracts (left panel). GST-Sir2 and GST-Fob1 associate with Nsi1 from whole cell extracts (right panel). (C) Western blots showing that the loss of Nsi1 decreases the amount of Sir2-Myc that coprecipitates with Fob1-TAP. Hexokinase was used as a loading control in all blots. The level of Sir2-Myc coprecipitated with Fob1-TAP in the presence or absence of Nsi1 was quantified and is shown to the right of the blots. Values represent the average of three independent experiments, and error bars indicate the SEM. Asterisk indicates P < 0.05, compared with wild-type cells (Student’s t-test).

Mentions: The association of Nsi1 with NTS1 and its involvement in NTS1 silencing raise the possibility that Nsi1 may physically interact with known rDNA silencing factors, such as Net1, Sir2 and Fob1. To determine whether Nsi1 interacts with Net1, Sir2 and Fob1, we performed a BiFC assay (26). We tagged NET1, SIR2 and FOB1 with VN in MATa cells and NSI1 with VC in MATα cells, and then generated diploid strains co-expressing VC-tagged Nsi1 and VN-tagged Net1, Sir2 or Fob1. When analyzed by fluorescence microscopy, all cells showed clear BiFC signals that overlapped exactly with the signal of Nop56-RFP, a well-characterized nucleolar marker (Supplementary Figure S5A). To confirm the interaction of Nsi1 with Net1, Sir2 and Fob1, we carried out CoIP experiments using cell extracts prepared from yeast strains co-expressing HA-Nsi1 and Net1-Myc, Sir2-Myc or Fob1-Myc. Immunoprecipitation of Net1-Myc, Sir2-Myc and Fob1-Myc all resulted in the coprecipitation of HA-Nsi1 (Figure 3A). We also tested the interaction of Nsi1 with Net1, Sir2 and Fob1 using a GST pull-down assay. We purified bacterially expressed GST and GST-Nsi1, incubated them with whole yeast cell extracts, and analyzed the bound fractions by western blotting. We found that GST-Nsi1 associated with Net1-HA, Sir2-GFP and Fob1-Myc, whereas the GST control did not (Figure 3B, left panel). We also observed that both GST-Sir2 and GST-Fob1 associated with GFP-Nsi1 (Figure 3B, right panel). Taken together, these results suggest that Nsi1 physically interacts with the Net1 and Sir2 subunits of the RENT complex and Fob1.Figure 3.


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

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

Nsi1 physically interacts with Net1, Sir2 and Fob1, and the loss of Nsi1 decreases the interaction between Fob1 and Sir2. (A) Western blots showing that HA-Nsi1 coprecipitates with Net1-Myc (left panel), Sir2-Myc (middle panel) and Fob1-Myc (right panel) from whole cell extracts. (B) GST pull-down assay showing direct association of Nsi1 with Net1, Sir2 and Fob1. GST-Nsi1 associates with Net1, Sir2 and Fob1 from whole cell extracts (left panel). GST-Sir2 and GST-Fob1 associate with Nsi1 from whole cell extracts (right panel). (C) Western blots showing that the loss of Nsi1 decreases the amount of Sir2-Myc that coprecipitates with Fob1-TAP. Hexokinase was used as a loading control in all blots. The level of Sir2-Myc coprecipitated with Fob1-TAP in the presence or absence of Nsi1 was quantified and is shown to the right of the blots. Values represent the average of three independent experiments, and error bars indicate the SEM. Asterisk indicates P < 0.05, compared with wild-type cells (Student’s t-test).
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Related In: Results  -  Collection

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gks188-F3: Nsi1 physically interacts with Net1, Sir2 and Fob1, and the loss of Nsi1 decreases the interaction between Fob1 and Sir2. (A) Western blots showing that HA-Nsi1 coprecipitates with Net1-Myc (left panel), Sir2-Myc (middle panel) and Fob1-Myc (right panel) from whole cell extracts. (B) GST pull-down assay showing direct association of Nsi1 with Net1, Sir2 and Fob1. GST-Nsi1 associates with Net1, Sir2 and Fob1 from whole cell extracts (left panel). GST-Sir2 and GST-Fob1 associate with Nsi1 from whole cell extracts (right panel). (C) Western blots showing that the loss of Nsi1 decreases the amount of Sir2-Myc that coprecipitates with Fob1-TAP. Hexokinase was used as a loading control in all blots. The level of Sir2-Myc coprecipitated with Fob1-TAP in the presence or absence of Nsi1 was quantified and is shown to the right of the blots. Values represent the average of three independent experiments, and error bars indicate the SEM. Asterisk indicates P < 0.05, compared with wild-type cells (Student’s t-test).
Mentions: The association of Nsi1 with NTS1 and its involvement in NTS1 silencing raise the possibility that Nsi1 may physically interact with known rDNA silencing factors, such as Net1, Sir2 and Fob1. To determine whether Nsi1 interacts with Net1, Sir2 and Fob1, we performed a BiFC assay (26). We tagged NET1, SIR2 and FOB1 with VN in MATa cells and NSI1 with VC in MATα cells, and then generated diploid strains co-expressing VC-tagged Nsi1 and VN-tagged Net1, Sir2 or Fob1. When analyzed by fluorescence microscopy, all cells showed clear BiFC signals that overlapped exactly with the signal of Nop56-RFP, a well-characterized nucleolar marker (Supplementary Figure S5A). To confirm the interaction of Nsi1 with Net1, Sir2 and Fob1, we carried out CoIP experiments using cell extracts prepared from yeast strains co-expressing HA-Nsi1 and Net1-Myc, Sir2-Myc or Fob1-Myc. Immunoprecipitation of Net1-Myc, Sir2-Myc and Fob1-Myc all resulted in the coprecipitation of HA-Nsi1 (Figure 3A). We also tested the interaction of Nsi1 with Net1, Sir2 and Fob1 using a GST pull-down assay. We purified bacterially expressed GST and GST-Nsi1, incubated them with whole yeast cell extracts, and analyzed the bound fractions by western blotting. We found that GST-Nsi1 associated with Net1-HA, Sir2-GFP and Fob1-Myc, whereas the GST control did not (Figure 3B, left panel). We also observed that both GST-Sir2 and GST-Fob1 associated with GFP-Nsi1 (Figure 3B, right panel). Taken together, these results suggest that Nsi1 physically interacts with the Net1 and Sir2 subunits of the RENT complex and Fob1.Figure 3.

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