RBF1, a Plant Homolog of the Bacterial Ribosome-Binding Factor RbfA, Acts in Processing of the Chloroplast 16S Ribosomal RNA

Fristedt, Rikard; Scharff, Lars B.; Clarke, Cornelia A.; Wang, Qin; Lin, Cheng‐Kuan; Merchant, Sabeeha; Bock, Ralph

doi:10.1104/pp.113.228338

Cited by 50 publications

(64 citation statements)

References 86 publications

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“…Figure 4B. The previously shown primer extension analysis in Figure 4B is correct and only replaced because an additional control mutant defective in 16S rRNA processing, rbf1-1 (Fristedt et al, 2014), is included in the analysis. Note the apparent extension of the "mature" 16S transcript in rap-1 compared with the wild-type and rbf1-1.…”

Section: Author Contributionsmentioning

confidence: 99%

“…Expected sizes of fragments were estimated from the running fronts of xylene cyanol (;40 nucleotides) and bromophenol blue (;15 nucleotides) indicated on the right. RNA gel blot analyses of small RNAs from the wild type, rap-1, and an additional control RNA from the Arabidopsis rbf1-1 mutant, described to also reveal a defect in 16S rRNA processing (Fristedt et al, 2014). Thirty micrograms of total leaf RNA was fractionated in denaturing polyacrylamide gels and transferred to a charged nylon membrane.…”

Section: Author Contributionsmentioning

confidence: 99%

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RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation

Kleinknecht¹,

Wang²,

Stübe³

et al. 2014

The Plant Cell

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The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

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Section: Author Contributionsmentioning

confidence: 99%

Section: Author Contributionsmentioning

confidence: 99%

RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation

Kleinknecht¹,

Wang²,

Stübe³

et al. 2014

The Plant Cell

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“…6C) similar to the effects seen for the rRNAs. Because no defect in the steady-state levels of the mature mRNAs was noticed, this is probably a secondary effect of the overall perturbation of the translation machinery (Fristedt et al, 2014). No processing defect was observed for psbA.…”

Section: Rh50 Is Involved In Plastid Rrna Metabolismmentioning

confidence: 85%

The DEAD-box RNA Helicase RH50 Is a 23S-4.5S rRNA Maturation Factor that Functionally Overlaps with the Plastid Signaling Factor GUN1

Paieri

Tadini

Manavski³

et al. 2017

Plant Physiol.

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DEAD-box RNA helicases (DBRHs) modulate RNA secondary structure, allowing RNA molecules to adopt the conformations required for interaction with their target proteins. RH50 is a chloroplast-located DBRH that colocalizes and is coexpressed with GUN1, a central factor in chloroplast-to-nucleus signaling. When combined with mutations that impair plastid gene expression (prors1-1, prpl11-1, prps1-1, prps21-1, prps17-1, and prpl24-1), rh50 and gun1 mutations evoke similar patterns of epistatic effects. These observations, together with the synergistic growth phenotype of the double mutant rh50-1 gun1-102, suggest that RH50 and GUN1 are functionally related and that this function is associated with plastid gene expression, in particular ribosome functioning. However, rh50-1 itself is not a gun mutant, although-like gun1-102-the rh50-1 mutation suppresses the downregulation of nuclear genes for photosynthesis induced by the prors1-1 mutation. The RH50 protein comigrates with ribosomal particles, and is required for efficient translation of plastid proteins. RH50 binds to transcripts of the 23S-4.5S intergenic region and, in its absence, levels of the corresponding rRNA processing intermediate are strongly increased, implying that RH50 is required for the maturation of the 23S and 4.5S rRNAs. This inference is supported by the finding that loss of RH50 renders chloroplast protein synthesis sensitive to erythromycin and exposure to cold. Based on these results, we conclude that RH50 is a plastid rRNA maturation factor.

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“…The free ribosomal fractions (<80S; lanes 1 to 6) and polysomal fractions (lanes 7 to 12) were determined using a puromycin control (bottom) as described in the text. 39-processed 16S rRNA from polysomal fractions has been reported previously in the mutant rbf1, which accumulates high levels of such molecules (Fristedt et al, 2014), and it is clear that mature 16S rRNA is the dominant polysome species in both the wild type and rnc3/4. Overall, this points toward unimpeded assembly of functional ribosomes in the absence of chloroplast Mini-III.…”

Section: Improperly Processed Rrna Precursors Are Incorporated Into Rmentioning

confidence: 85%

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

et al. 2015

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RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 39 end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 39 extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

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RBF1, a Plant Homolog of the Bacterial Ribosome-Binding Factor RbfA, Acts in Processing of the Chloroplast 16S Ribosomal RNA

Cited by 50 publications

References 86 publications

RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation

RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation

The DEAD-box RNA Helicase RH50 Is a 23S-4.5S rRNA Maturation Factor that Functionally Overlaps with the Plastid Signaling Factor GUN1

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

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