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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

Gel mobility shift assay to detect RNA binding activity of NSN1. (A) Maltose-binding protein (MBP)-fused NSN1 and NSN1 derivatives were purified from E. coli, and the eluted proteins were visualized by Coomassie blue staining. NSN1-N, N-terminal domain of NSN1 (amino acids 1–174); RBD, a putative RNA-binding domain of NSN1 (amino acids 374–400). Size markers are indicated. (B, C) MBP, MBP:NSN1, MBP:NSN1-N, and MBP:RBD fusion proteins (100 pmol) were incubated with 200ng of radiolabelled 25S rRNA (B) or 18S rRNA (C) with or without GTP (100 μM). Bound (B) and unbound (U) RNAs were resolved on an agarose gel and visualized by phosphorimaging.
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Figure 3: Gel mobility shift assay to detect RNA binding activity of NSN1. (A) Maltose-binding protein (MBP)-fused NSN1 and NSN1 derivatives were purified from E. coli, and the eluted proteins were visualized by Coomassie blue staining. NSN1-N, N-terminal domain of NSN1 (amino acids 1–174); RBD, a putative RNA-binding domain of NSN1 (amino acids 374–400). Size markers are indicated. (B, C) MBP, MBP:NSN1, MBP:NSN1-N, and MBP:RBD fusion proteins (100 pmol) were incubated with 200ng of radiolabelled 25S rRNA (B) or 18S rRNA (C) with or without GTP (100 μM). Bound (B) and unbound (U) RNAs were resolved on an agarose gel and visualized by phosphorimaging.

Mentions: It has been reported that the ribosome assembly GTPases contain RNA-binding domains in addition to the GTPase domain; yeast Nug1 binds directly to 5S rRNA and tRNA via its N-terminal domain (Bassler et al., 2006; Karbstein, 2007). To determine if NSN1 binds RNA, recombinant proteins of full-length NSN1 and the NSN1 N-terminal domain (NSN1-N; amino acids 1–174) fused to MBP were prepared. The RNA binding activity of a region (residues 374–400) that was previously annotated as a putative RNA-binding domain (RBD; Wang et al., 2012a) was also tested. The corresponding cDNA fragments were cloned into the pMAL vector, expressed in Escherichia coli, and recombinant MBP fusion proteins were affinity-purified using the N-terminal MBP tag (Fig. 3A). The binding of these recombinant proteins to 25S rRNA as an RNA substrate was determined using EMSAs. 32P-labeled 25S rRNA synthesized in vitro was incubated with MBP:NSN1, MBP:NSN1-N, MBP:RBD, and MBP in the absence or presence of 100 μM GTP, and RNA–protein complexes were resolved by agarose gel electrophoresis. Both MBP:NSN1 and MBP:NSN1-N readily formed stable RNA–protein complexes regardless of the GTP status, whereas neither MBP:RBD nor MBP complexed with RNA (Fig. 3B). MBP:NSN1 and MBP:NSN1-N also formed RNA–protein complexes with 32P-labelled 18S rRNA with or without GTP, whereas MBP:RBD or MBP did not (Fig. 3C). These results indicate that NSN1 has an RNA-binding activity, and the NSN1 N-terminal domain contributes to the binding.


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)

Gel mobility shift assay to detect RNA binding activity of NSN1. (A) Maltose-binding protein (MBP)-fused NSN1 and NSN1 derivatives were purified from E. coli, and the eluted proteins were visualized by Coomassie blue staining. NSN1-N, N-terminal domain of NSN1 (amino acids 1–174); RBD, a putative RNA-binding domain of NSN1 (amino acids 374–400). Size markers are indicated. (B, C) MBP, MBP:NSN1, MBP:NSN1-N, and MBP:RBD fusion proteins (100 pmol) were incubated with 200ng of radiolabelled 25S rRNA (B) or 18S rRNA (C) with or without GTP (100 μM). Bound (B) and unbound (U) RNAs were resolved on an agarose gel and visualized by phosphorimaging.
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Figure 3: Gel mobility shift assay to detect RNA binding activity of NSN1. (A) Maltose-binding protein (MBP)-fused NSN1 and NSN1 derivatives were purified from E. coli, and the eluted proteins were visualized by Coomassie blue staining. NSN1-N, N-terminal domain of NSN1 (amino acids 1–174); RBD, a putative RNA-binding domain of NSN1 (amino acids 374–400). Size markers are indicated. (B, C) MBP, MBP:NSN1, MBP:NSN1-N, and MBP:RBD fusion proteins (100 pmol) were incubated with 200ng of radiolabelled 25S rRNA (B) or 18S rRNA (C) with or without GTP (100 μM). Bound (B) and unbound (U) RNAs were resolved on an agarose gel and visualized by phosphorimaging.
Mentions: It has been reported that the ribosome assembly GTPases contain RNA-binding domains in addition to the GTPase domain; yeast Nug1 binds directly to 5S rRNA and tRNA via its N-terminal domain (Bassler et al., 2006; Karbstein, 2007). To determine if NSN1 binds RNA, recombinant proteins of full-length NSN1 and the NSN1 N-terminal domain (NSN1-N; amino acids 1–174) fused to MBP were prepared. The RNA binding activity of a region (residues 374–400) that was previously annotated as a putative RNA-binding domain (RBD; Wang et al., 2012a) was also tested. The corresponding cDNA fragments were cloned into the pMAL vector, expressed in Escherichia coli, and recombinant MBP fusion proteins were affinity-purified using the N-terminal MBP tag (Fig. 3A). The binding of these recombinant proteins to 25S rRNA as an RNA substrate was determined using EMSAs. 32P-labeled 25S rRNA synthesized in vitro was incubated with MBP:NSN1, MBP:NSN1-N, MBP:RBD, and MBP in the absence or presence of 100 μM GTP, and RNA–protein complexes were resolved by agarose gel electrophoresis. Both MBP:NSN1 and MBP:NSN1-N readily formed stable RNA–protein complexes regardless of the GTP status, whereas neither MBP:RBD nor MBP complexed with RNA (Fig. 3B). MBP:NSN1 and MBP:NSN1-N also formed RNA–protein complexes with 32P-labelled 18S rRNA with or without GTP, whereas MBP:RBD or MBP did not (Fig. 3C). These results indicate that NSN1 has an RNA-binding activity, and the NSN1 N-terminal domain contributes to the binding.

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