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

Highlight Plant NSN1 has GTPase and RNA-binding activities. In the nucleolus, NSN1 is involved in rRNA maturation and ribosome biogenesis through interaction with PES, EBP2, and several ribosomal proteins.


Agrobacterium-mediated transient expression
Agroinfiltration was carried out as described previously (Voinnet et al., 2003) et al., 2003) was co-infiltrated to achieve maximum levels of protein expression. Expressed proteins were analyzed at 48h post-infiltration.

Real-time quantitative RT-PCR
Real-time quantitative PCR was carried out using gene-specific primers as described previously (Cho et al., 2013).

Bimolecular fluorescence complementation (BiFC)
The cDNAs of EBP2, PES, and various ribosomal protein genes of Arabidopsis were PCRamplified and cloned into the pSPYNE vector containing the N-terminal region of YFP (YFP N ; amino acid residues 1-155). Similarly, the coding region of NSN1 and PES were cloned into pSPYCE vector containing the C-terminal region of YFP (YFP C ; residues 156-239). Various combinations of these pSPYNE and pSPYCE fusion constructs were agroinfiltrated together into the leaves of 3-week-old N.benthamiana plants as described (Cho et al., 2013). After 48 h, protoplasts were generated and YFP signal was detected using a confocal laser scanning microsope (Zeiss LSM510).

Measurement of in vivo H 2 O 2 levels
NBT staining was carried out as described previously (Lee et al., 2013). H 2 DCFDA staining was carried out as described previously (Ahn et al., 2011).

Purification of recombinant proteins
To purify recombinant proteins of NSN1 and its mutants for GTPase assays, the corresponding NSN1 cDNA fragments were PCR-amplified and cloned into the pMAL TM c2 vector (New England Biolabs). The MBP fusion proteins were purified using amylose resin following the manufacturer's instructions (New England Biolabs). Purified NSN1 protein and its variants were concentrated using Amicon Ultra Centrifugal Filters (Millipore). To purify MBP:NSN1-N and MBP:RBD for RNA-binding assays, the NSN1 cDNA fragments corresponding to amino acid residues 1-174 and 374-400, respectively, were PCR-amplified and cloned into the pMAL TM c2 vector, and the recombinant proteins were purified as described above.

GTPase assay
The turnover rate (k cat ) of recombinant proteins of NSN1 was measured as described previously (Im et al., 2011;Jeon et al., 2014). A reaction mixture containing 3 μM

Statistical analyses
Two-tailed Student's t-tests were performed using the Minitab 16 program (Minitab Inc.; http://www.minitab.com/en-KR/default.aspx) to investigate the statistical differences between the responses of the samples. Significant differences between control and other samples were indicated by one (P ≤ 0.05) or two (P ≤0.01) asterisks.