Limits...
The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis.

Jeon Y, Park YJ, Cho HK, Jung HJ, Ahn TK, Kang H, Pai HS - J. Exp. Bot. (2015)

Bottom Line: Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation.NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression.Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

No MeSH data available.


Related in: MedlinePlus

Analyses of protein interactions of NSN1. (A) BiFC analyses of NSN1 interaction with PES and EBP2. Proteins were co-expressed as either YFPN (YN) or YFPC (YC) fusion proteins in N. benthamiana leaves using agroinfiltration. Protoplasts were prepared from the infiltrated leaves and observed for YFP fluorescence after brief staining with DAPI to visualize nuclei. (B) BiFC analyses of NSN1 interaction with ribosomal proteins. Left, confocal microscopy; right, immunoblot analysis (IB). (C, D) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves co-expressing PES:Flag and NSN1:HA (C) or Myc:EBP2 and NSN1:HA (D) fusion proteins. Extracts were subjected to immunoprecipitation (IP) with anti-HA antibody, and co-immunoprecipitated proteins were detected by immunoblotting with anti-Flag and anti-Myc antibodies. (E) VIGS phenotypes of EBP2 using three different EBP2 constructs in N. benthamiana at 14 DAI. (F) Real-time quantitative RT–PCR analysis of EBP2 transcript levels in TRV:EBP2(N) and TRV:EBP2(C) plants (14 DAI) using EBP2-A and EBP2-B primers. The fourth leaf above the infiltrated leaf was used for the analysis. The β-tubulin mRNA level was used as control. Data represent the mean ±SD of three replicates per experiment; *P≤0.05; **P≤0.01. (This figure is available in colour at JXB online.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4588883&req=5

Figure 4: Analyses of protein interactions of NSN1. (A) BiFC analyses of NSN1 interaction with PES and EBP2. Proteins were co-expressed as either YFPN (YN) or YFPC (YC) fusion proteins in N. benthamiana leaves using agroinfiltration. Protoplasts were prepared from the infiltrated leaves and observed for YFP fluorescence after brief staining with DAPI to visualize nuclei. (B) BiFC analyses of NSN1 interaction with ribosomal proteins. Left, confocal microscopy; right, immunoblot analysis (IB). (C, D) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves co-expressing PES:Flag and NSN1:HA (C) or Myc:EBP2 and NSN1:HA (D) fusion proteins. Extracts were subjected to immunoprecipitation (IP) with anti-HA antibody, and co-immunoprecipitated proteins were detected by immunoblotting with anti-Flag and anti-Myc antibodies. (E) VIGS phenotypes of EBP2 using three different EBP2 constructs in N. benthamiana at 14 DAI. (F) Real-time quantitative RT–PCR analysis of EBP2 transcript levels in TRV:EBP2(N) and TRV:EBP2(C) plants (14 DAI) using EBP2-A and EBP2-B primers. The fourth leaf above the infiltrated leaf was used for the analysis. The β-tubulin mRNA level was used as control. Data represent the mean ±SD of three replicates per experiment; *P≤0.05; **P≤0.01. (This figure is available in colour at JXB online.)

Mentions: Mammalian NS forms a large protein complex that contains Pescadillo, EBP2, DDX21, and a subset of ribosomal proteins (Romanova et al., 2009). BiFC was used to test if NSN1 interacts with Arabidopsis Pescadillo (PES) and EBP2 in vivo using (Fig. 4A). BiFC has been widely used for visualization of protein–protein interactions in living cells (Walter et al., 2004). Plant PES is a nucleolar protein that plays a crucial role in biogenesis of the 60S ribosomal large subunit through a functional link with BOP1 and WDR12 (Cho et al., 2013). Combinations of proteins were co-expressed as N- and a C-terminal yellow fluorescent protein fusion proteins (YFPN and YFPC) in N. benthamiana leaves by agroinfiltration. After 48h, protoplasts were prepared from the infiltrated leaves and observed with confocal laser scanning microscopy. All combinations of fusion protein expression resulted in strong nucleolar YFP fluorescence, indicating that NSN1, PES, and EBP2 interact with each other in the nucleolus (Fig. 4A). The N-terminal domain of NSN1 (NSN1-N; amino acids 1–174) was sufficient for nucleolar interaction with PES and EBP2, whereas the deletion of the NSN1 N-terminal 94 amino acids (∆N) caused protein interactions in both the nucleolus and the nucleoplasm (Supplementary Fig. S6 at JXB online). A deletion of the GTP-binding motifs (∆GD) or the NSN1 C-terminal domain (∆C) did not affect the protein interactions in the nucleolus. These results suggest that the NSN1 N-terminal domain plays a role in the interactions of NSN1 with PES and EBP2, but other domains of NSN1 also contribute to the protein interactions. Next, BiFC interactions between NSN1 and PES mutants were examined (Supplementary Fig. S7). NSN1 interacted with the N-terminal PES-N domain in the nucleolus, but with ∆PES-N (a PES mutant lacking the PES-N domain) in the nucleus. Previously, GFP-fused ∆PES-N exhibited a diffuse nucleoplasmic distribution (Cho et al., 2013). These results suggest that PES interaction with NSN1 involves the PES-N domain as well as other regions of PES. BiFC analyses also revealed an interaction of NSN1 with ribosomal proteins RPL13, RPL14, and RPS6, but not with RPL11 (Fig. 4B, left), despite the high expression of both NSN1:YFPC and RPL11:YFPN in the infiltrated leaves (Fig. 4B, right).


The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis.

Jeon Y, Park YJ, Cho HK, Jung HJ, Ahn TK, Kang H, Pai HS - J. Exp. Bot. (2015)

Analyses of protein interactions of NSN1. (A) BiFC analyses of NSN1 interaction with PES and EBP2. Proteins were co-expressed as either YFPN (YN) or YFPC (YC) fusion proteins in N. benthamiana leaves using agroinfiltration. Protoplasts were prepared from the infiltrated leaves and observed for YFP fluorescence after brief staining with DAPI to visualize nuclei. (B) BiFC analyses of NSN1 interaction with ribosomal proteins. Left, confocal microscopy; right, immunoblot analysis (IB). (C, D) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves co-expressing PES:Flag and NSN1:HA (C) or Myc:EBP2 and NSN1:HA (D) fusion proteins. Extracts were subjected to immunoprecipitation (IP) with anti-HA antibody, and co-immunoprecipitated proteins were detected by immunoblotting with anti-Flag and anti-Myc antibodies. (E) VIGS phenotypes of EBP2 using three different EBP2 constructs in N. benthamiana at 14 DAI. (F) Real-time quantitative RT–PCR analysis of EBP2 transcript levels in TRV:EBP2(N) and TRV:EBP2(C) plants (14 DAI) using EBP2-A and EBP2-B primers. The fourth leaf above the infiltrated leaf was used for the analysis. The β-tubulin mRNA level was used as control. Data represent the mean ±SD of three replicates per experiment; *P≤0.05; **P≤0.01. (This figure is available in colour at JXB online.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4588883&req=5

Figure 4: Analyses of protein interactions of NSN1. (A) BiFC analyses of NSN1 interaction with PES and EBP2. Proteins were co-expressed as either YFPN (YN) or YFPC (YC) fusion proteins in N. benthamiana leaves using agroinfiltration. Protoplasts were prepared from the infiltrated leaves and observed for YFP fluorescence after brief staining with DAPI to visualize nuclei. (B) BiFC analyses of NSN1 interaction with ribosomal proteins. Left, confocal microscopy; right, immunoblot analysis (IB). (C, D) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves co-expressing PES:Flag and NSN1:HA (C) or Myc:EBP2 and NSN1:HA (D) fusion proteins. Extracts were subjected to immunoprecipitation (IP) with anti-HA antibody, and co-immunoprecipitated proteins were detected by immunoblotting with anti-Flag and anti-Myc antibodies. (E) VIGS phenotypes of EBP2 using three different EBP2 constructs in N. benthamiana at 14 DAI. (F) Real-time quantitative RT–PCR analysis of EBP2 transcript levels in TRV:EBP2(N) and TRV:EBP2(C) plants (14 DAI) using EBP2-A and EBP2-B primers. The fourth leaf above the infiltrated leaf was used for the analysis. The β-tubulin mRNA level was used as control. Data represent the mean ±SD of three replicates per experiment; *P≤0.05; **P≤0.01. (This figure is available in colour at JXB online.)
Mentions: Mammalian NS forms a large protein complex that contains Pescadillo, EBP2, DDX21, and a subset of ribosomal proteins (Romanova et al., 2009). BiFC was used to test if NSN1 interacts with Arabidopsis Pescadillo (PES) and EBP2 in vivo using (Fig. 4A). BiFC has been widely used for visualization of protein–protein interactions in living cells (Walter et al., 2004). Plant PES is a nucleolar protein that plays a crucial role in biogenesis of the 60S ribosomal large subunit through a functional link with BOP1 and WDR12 (Cho et al., 2013). Combinations of proteins were co-expressed as N- and a C-terminal yellow fluorescent protein fusion proteins (YFPN and YFPC) in N. benthamiana leaves by agroinfiltration. After 48h, protoplasts were prepared from the infiltrated leaves and observed with confocal laser scanning microscopy. All combinations of fusion protein expression resulted in strong nucleolar YFP fluorescence, indicating that NSN1, PES, and EBP2 interact with each other in the nucleolus (Fig. 4A). The N-terminal domain of NSN1 (NSN1-N; amino acids 1–174) was sufficient for nucleolar interaction with PES and EBP2, whereas the deletion of the NSN1 N-terminal 94 amino acids (∆N) caused protein interactions in both the nucleolus and the nucleoplasm (Supplementary Fig. S6 at JXB online). A deletion of the GTP-binding motifs (∆GD) or the NSN1 C-terminal domain (∆C) did not affect the protein interactions in the nucleolus. These results suggest that the NSN1 N-terminal domain plays a role in the interactions of NSN1 with PES and EBP2, but other domains of NSN1 also contribute to the protein interactions. Next, BiFC interactions between NSN1 and PES mutants were examined (Supplementary Fig. S7). NSN1 interacted with the N-terminal PES-N domain in the nucleolus, but with ∆PES-N (a PES mutant lacking the PES-N domain) in the nucleus. Previously, GFP-fused ∆PES-N exhibited a diffuse nucleoplasmic distribution (Cho et al., 2013). These results suggest that PES interaction with NSN1 involves the PES-N domain as well as other regions of PES. BiFC analyses also revealed an interaction of NSN1 with ribosomal proteins RPL13, RPL14, and RPS6, but not with RPL11 (Fig. 4B, left), despite the high expression of both NSN1:YFPC and RPL11:YFPN in the infiltrated leaves (Fig. 4B, right).

Bottom Line: Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation.NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression.Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

No MeSH data available.


Related in: MedlinePlus