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PRBP plays a role in plastid ribosomal RNA maturation and chloroplast biogenesis in Nicotiana benthamiana

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Abstract

In the present study, we investigated protein characteristics and physiological functions of PRBP (plastid RNA-binding protein) in Nicotiana benthamiana. PRBP fused to green fluorescent protein (GFP) localized to the chloroplasts. Recombinant PRBP proteins bind to single-stranded RNA in vitro, but not to DNA in a double- or a single-stranded form. Virus-induced gene silencing (VIGS) of PRBP resulted in leaf yellowing in N. benthamiana. At the cellular level, PRBP depletion disrupted chloroplast biogenesis: chloroplast number and size were reduced, and the thylakoid membrane was poorly developed. In PRBP-silenced leaves, protein levels of plastid-encoded genes were significantly reduced, whereas their mRNA levels were normal regardless of their promoter types indicating that PRBP deficiency primarily affects translational or post-translational processes. Depletion of PRBP impaired processing of the plastid-encoded 4.5S ribosomal RNA, resulting in accumulation of the larger precursor rRNAs in the chloroplasts. In addition, PRBP-deficient chloroplasts contained significantly reduced levels of mature 4.5S and 5S rRNAs in the polysomal fractions, indicating decreased chloroplast translation. These results suggest that PRBP plays a role in chloroplast rRNA processing and chloroplast development in higher plants.

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Acknowledgments

This research was supported by Mid-career Researcher Program (No. 20100026168 and No. 20100000314) and Plant Signaling Network Research Center (No. 2010-0001462), both of which are funded by National Research Foundation of Korea.

Author information

Correspondence to Hyun-Sook Pai.

Additional information

GenBank accession number: HM236482.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 17 KB)

SupplementalFig.1. Sequence alignment of PRBP proteins. a The PRBP protein structure showing the N-terminal transit peptide (TP). b Amino acid sequences of PRBP and its homologs from tobacco (PRBP-Nt), tomato (PRBP-Le), and Arabidopsis (PRBP-At) were aligned. The residues that are conserved between the sequences are boxed in black or light gray based on the degree of conservation. The overline indicates the transit peptide. c Amino acid sequences of the N-termini of PRBP, PRBP-At, and the putative organellar DEAD-box RNA helicases of Arabidopsis (At2g07750 and At1g63250) were aligned (PDF 48 KB)

425_2011_1362_MOESM3_ESM.pdf

SupplementalFig.2. Expression profiles of Arabidopsis PRBP (At2g37920) in diverse tissues based on Genevestigator analysis (http://www.genevestigator.com/). Expression is expressed relative to the transcript level in roots (PDF 116 KB)

SupplementalFig.3. RNA gel blot analysis using the 163-nucleotide probe. Positions of the PCR-amplified probes (probes A and B) are marked by thick lines under the rrn operon. The size of the ~3.2 kb 23S–4.5S precursor and the ~2.9 kb mature 23S rRNA is shown underneath as thin lines. The RNA gel blot shown in Fig. 7c was hybridized with the probe B containing the 163-nucleotide region between the tRNA-Ala (trnA) mature 3′-end and the 23S rRNA mature 5′-end. The probe B recognized only the ~3.2 kb 23S–4.5S precursor (PDF 54 KB)

SupplementalFig.4. PRBP binding to the 23S–4.5S rRNA precursor Increasing concentrations of His:PRBP fusion protein (0, 10, 50, and 100 pmol) were incubated with two different radiolabeled ssRNA probes (20 pM) derived from the 23S–4.5S rRNA precursor sequence. Bound (B) and unbound (U) RNAs were resolved on a native polyacrylamide gel. Only part of the N. benthamiana chloroplast rrn operon is presented. Positions of the ssRNA probes (probes A and B) are marked by thick lines under the operon. The probes contain all or part of the 163-nucleotide region between the 23S rRNA mature 3′-end and the 4.5S rRNA mature 5′-end in the rrn operon (PDF 38 KB)

SupplementalFig.5. Nucleolar pre-rRNA processing. a Structure of the primary 35S pre-rRNA. The mature 18S, 5.8S, and 25S rRNA sequences are flanked by the external transcribed sequences 5′-ETS and 3′-ETS and separated by internal transcribed spacers ITS1 and ITS2. Positions of probes in the 35S rRNA primary transcript are marked by the thick lines. b RNA gel blot analysis was performed with total RNA isolated from the leaves of the TRV, TRV:PRBP(N), and TRV:PRBP(C) lines. The RNA gel blots (15 μg total RNA per lane) were hybridized with probes for 18S, 5.8S, and 25S rRNAs. An ethidium bromide (EtBr)-stained gel shows total ribosomal RNA. c RNA gel blot analysis was performed with total RNA isolated from TRV and TRV:PRBP(N) leaves using end-labeled ITS1 and ITS2 probes. d Absorbance profile of ribosomes at 254 nm. Ribosomes were purified from leaves of TRV and TRV:PRBP(N) lines by sucrose density gradient ultracentrifugation. Positions of the ribosome small subunits (40S), large subunits and monosomes (60S/80S), and polysomes are marked (PDF 245 KB)

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Park, Y., Cho, H., Jung, H.J. et al. PRBP plays a role in plastid ribosomal RNA maturation and chloroplast biogenesis in Nicotiana benthamiana . Planta 233, 1073–1085 (2011). https://doi.org/10.1007/s00425-011-1362-7

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Keywords

  • Chloroplast abnormality
  • Chloroplast ribosomal RNA
  • Chloroplast targeting
  • RNA binding
  • Virus-induced gene silencing