AtRsgA from Arabidopsis thaliana controls maturation of the small subunit of the chloroplast ribosome

SummaryPlastid ribosomes are very similar in structure and function to ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favourable at biological conditions, it requires activity of many assembly factors. Here, we have characterized a homolog of bacterial rsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA-sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were upregulated, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signalling pathways. To conclude, this study reveals a new chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.Significance statementAtRsgA is an assembly factor necessary for maturation of the small subunit of the chloroplast ribosome. Depletion of AtRsgA leads to dwarfed, chlorotic plants and smaller, but more numerous chloroplasts. Large-scale transcriptomic and proteomic analysis revealed that chloroplast-encoded and - targeted proteins were less abundant, while the corresponding transcripts were upregulated in the mutant. We analyse the transcriptional responses of several retrograde signalling pathways to suggest a mechanism underlying this compensatory response.

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Proteomic analysis of haem-binding protein from Arabidopsis thaliana and Cyanidioschyzon merolae

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2019.0488 ◽

2020 ◽

Vol 375 (1801) ◽

pp. 20190488 ◽

Cited By ~ 1

Author(s):

Takayuki Shimizu ◽

Rintaro Yasuda ◽

Yui Mukai ◽

Ryo Tanoue ◽

Tomohiro Shimada ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Proteomic Analysis ◽

High Performance ◽

Biochemical Analysis ◽

Cyanidioschyzon Merolae ◽

Genes Expression ◽

Retrograde Signalling ◽

Coordinated Expression ◽

Retrograde Signal ◽

Affinity Beads

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae . Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

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Ribosome biogenesis factor Ltv1 chaperones the assembly of the small subunit head

The Journal of Cell Biology ◽

10.1083/jcb.201804163 ◽

2018 ◽

Vol 217 (12) ◽

pp. 4141-4154 ◽

Cited By ~ 11

Author(s):

Jason C. Collins ◽

Homa Ghalei ◽

Joanne R. Doherty ◽

Haina Huang ◽

Rebecca N. Culver ◽

...

Keyword(s):

Cancer Cells ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Breast Cancer Cells ◽

Ribosomal Subunit ◽

Small Subunit ◽

Yeast Genetics ◽

Structure Probing ◽

Rna Quality Control ◽

Assembly Factor

The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.

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Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0392 ◽

2017 ◽

Vol 372 (1730) ◽

pp. 20160392 ◽

Cited By ~ 10

Author(s):

Barbara Karpinska ◽

Sarah Owdah Alomrani ◽

Christine H. Foyer

Keyword(s):

Arabidopsis Thaliana ◽

Redox State ◽

Guard Cells ◽

Signalling Pathways ◽

Mrna Levels ◽

Cellular Redox ◽

Mitochondrial Inhibitors ◽

Retrograde Signalling ◽

Stomatal Guard Cells ◽

Cellular Redox State

Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction–oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1 , WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

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Faculty Opinions recommendation of Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1007601.96055 ◽

2002 ◽

Author(s):

Paul Dupree

Keyword(s):

Arabidopsis Thaliana ◽

Glucose Transporter ◽

Functional Characterization

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The small subunit processome in ribosome biogenesis-progress and prospects

Wiley Interdisciplinary Reviews - RNA ◽

10.1002/wrna.57 ◽

2010 ◽

Vol 2 (1) ◽

pp. 1-21 ◽

Cited By ~ 106

Author(s):

Kathleen R. Phipps ◽

J. Michael Charette ◽

Susan J. Baserga

Keyword(s):

Ribosome Biogenesis ◽

Small Subunit

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The Small Subunit Processome Is Required for Cell Cycle Progression at G1

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0515 ◽

2004 ◽

Vol 15 (11) ◽

pp. 5038-5046 ◽

Cited By ~ 44

Author(s):

Kara A. Bernstein ◽

Susan J. Baserga

Keyword(s):

Cell Cycle ◽

Ribosome Biogenesis ◽

Cell Cycle Progression ◽

Small Subunit ◽

Cellular Growth ◽

Yeast Cells ◽

G1 Phase ◽

Relay Cell ◽

Ssu Processome ◽

Nucleolar Rna

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

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Mapping and Functional Characterization of the TAF11 Interaction with TFIIA

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.945-957.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 945-957 ◽

Cited By ~ 19

Author(s):

M. M. Robinson ◽

G. Yatherajam ◽

R. T. Ranallo ◽

A. Bric ◽

M. R. Paule ◽

...

Keyword(s):

Functional Characterization ◽

Regulation Of Gene Expression ◽

Small Subunit ◽

Molecular Organization ◽

Functional Link ◽

Essential Information ◽

Histone Fold ◽

Histone Fold Domain ◽

Promoter Dna

ABSTRACT TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

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