DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants

Chloroplast-localized DER (Double Era-like GTPase) contains two consecutive GTP-binding domains, each of which possesses GTPase activity. DER binds to 23S and 16S ribosomal RNAs, and plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants


RNA gel blot analysis
For RNA gel blot analysis, total RNA was prepared with TRIzol TM Reagent (Gibco-BRL) following the manufacturer's instructions. RNA gel blot analyses were performed with approximately 20 g total RNA as described previously (Jeon et al., 2012). To generate probes, cDNAs were PCR-amplified using published sequences and cloned for sequence verification. Probes were labeled with a DecaLabel DNA Labeling Kit (Thermo Scientific).

DAPI staining
DAPI staining and detection by confocal laser scanning microscopy was performed as described in Cho et al. (2004).

Confocal microscopy for subcellular localization of NbDER
NbDER cDNAs corresponding to the full-length coding region (Met-1 to Ala-651) and the N-

Transmission electron microscopy
Cotyledons and leaves were fixed with 2.5% (v/v) glutaraldehyde and with 1% osmium tetraoxide, followed by dehydration through an ethanol series, and embedded in Spurr's resin (EM Sciences, USA). Thin sections were prepared with a LKB III ultramicrotome and stained sequentially with 5% uranyl acetate and 3% lead citrate, and observed under a JEOL 1200 EXII transmission electron microscope.

Purification of recombinant proteins
To purify recombinant proteins of NbDER and its mutants for GTPase assays, the corresponding NbDER 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 proteins of MBP:NbDER, MBP:PM1, MBP:PM2, and MBP:PM1/2 were concentrated using Amicon Ultra Centrifugal Filters (Millipore). To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520651 was PCR-amplified and cloned into the pMAL TM c2 vector (New England Biolabs).

RNA binding assay
To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector. The constructs were digested with BamHI restriction enzyme, and RNAs were prepared by in vitro transcription using T7 RNA polymerase (Promega). For RNA binding assays, the RNA substrates were incubated with the purified recombinant MBP fusion proteins in binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 7.4% glycerol) on ice for 30 min. The reaction mixtures were loaded on 0.8% agarose gel, and RNA bands were visualized by UV light after ethidium bromide staining or by PhosphorImager (GE Healthcare Life Sciences). For EMSA competition assays, 25 picomoles of the recombinant proteins were incubated with variable ratios of radiolabeled (200 ng) and unlabeled RNAs, ranging from 1:0 to 1:20. A sequence-nonspecific 33 P-labeled RNA substrate (~160 nucleotides in length) was prepared by transcribing BamHI-digested pET-22b(+) plasmid using T7 RNA polymerase as described previously (Jeon et al., 2012).
For RNA-protein interaction analysis, the labeled sequence-nonspecific RNAs (30 ng) were incubated with purified recombinant proteins in binding buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, and 7% glycerol) for 30 min on ice. The mixture was separated on 6% non-denaturing polyacrylamide gel, and RNA bands were detected by PhosphorImager.

GTPase assay
The turnover rate (k cat ) of recombinant proteins of NbDER and its mutants was measured as described previously (Im et al., 2011). A reaction mixture containing 3 μM recombinant proteins and 1 mM GTP in GTPase assay buffer (20 mM Hepes, pH 8, 1 mM MgCl 4 , 0.5 mM DTT, and 1 mM NaN 3 ) was incubated at room temperature for 18 h. The released phosphate was quantified using the Biomol green reagent (Biomol Research Laboratories) according to the manufacturer's protocol. The catalytic constant was derived from the equation k cat = V max /C recombinant protein .

Sucrose density gradient analysis
GFP-fusion proteins of NbDER and its variants were expressed in N. benthamiana leaves by agroinfiltration, and the leaf extracts were fractionated through 15%-55% sucrose density gradients as described previously (Barkan 1993;Williams and Barkan, 2003). Proteins extracted from the fractions were separated by SDS-PAGE and subjected to immunoblotting with anti-GFP antibodies (Clontech) and anti-RPL10 antibodies (Santa Cruz Biotechnology).