Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes

Gawroński, Piotr; Jensen, Poul Erik; Karpiński, Stanisław; Leister, Dario; Scharff, Lars B.

doi:10.1104/pp.17.01564

Cited by 43 publications

(36 citation statements)

References 82 publications

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“…In E. coli, there is extensive mRNA-based, autogenous regulation of ribosomal proteins that control ribosome assembly, where ribosomal proteins bind their own mRNA to prevent translation (27). In light of reports that local mRNA structure and cotranslational events can hinder translation elongation (28)(29)(30), the observed increases in PS in ribosomal genes in Synechocystis may be at sites of autogenous or small RNA (sRNA) binding.…”

Section: Resultsmentioning

confidence: 99%

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

et al. 2018

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Cyanobacteria experience both rapid and periodic fluctuations in light and inorganic carbon (Ci) and have evolved regulatory mechanisms to respond to these, including extensive posttranscriptional gene regulation. We report the first genome-wide ribosome profiling data set for cyanobacteria, where ribosome occupancy on mRNA is quantified with codon-level precision. We measured the transcriptome and translatome of Synechocystis during autotrophic growth before (high carbon [HC] condition) and 24 h after removing CO2 from the feedgas (low carbon [LC] condition). Ribosome occupancy patterns in the 5′ untranslated region suggest that ribosomes can assemble there and slide to the Shine-Dalgarno site, where they pause. At LC, total translation was reduced by 80% and ribosome pausing was increased at stop and start codons and in untranslated regions, which may be a sequestration mechanism to inactivate ribosomes in response to rapid Ci depletion. Several stress response genes, such as thioredoxin M (sll1057), a putative endonuclease (slr0915), protease HtrA (slr1204), and heat shock protein HspA (sll1514) showed marked increases in translational efficiency at LC, indicating translational control in response to Ci depletion. Ribosome pause scores within open reading frames were mostly constant, though several ribosomal proteins had significantly altered pause score distributions at LC, which might indicate translational regulation of ribosome biosynthesis in response to Ci depletion. We show that ribosome profiling is a powerful tool to decipher dynamic gene regulation strategies in cyanobacteria. IMPORTANCE Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

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Section: Resultsmentioning

confidence: 99%

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

et al. 2018

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“…This mechanism was also shown to work in synthetic riboswitches in chloroplasts [16]. Furthermore, plastid mRNA secondary structure of coding regions was proposed to influence ribosome pausing [17].…”

Section: Introductionmentioning

confidence: 93%

Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Gawroński

Pałac

Scharff

2020

Plants

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mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

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“…mRNA structure has been shown to have an influence on translation slowdown [59,58], and a recent study implicated its role in pausing of chloroplast ribosomes [23]. Gawroński et al observed increased stability of mRNA secondary structure 31 nucleotides downstream of pausing site, for a sample of 78 stall sites.…”

Section: Proline Glycine and Negatively Charged Amino Acids Are Consmentioning

confidence: 99%

“…In a recent study, a significant pausing signal was observed downstream of the start of transmembrane domains (TMs) in chloroplasts. It occurred 52 amino acids downstream of the start of type II TMs and 34 amino acids for type I TMs [23]. The authors speculated that this would leave the time for the TMs to fold before translation would proceed.…”

Section: Little Evidence For Involvement Of Csss In Membrane Targetinmentioning

confidence: 99%

“…proline) in the P-and A-site attenuating the rates of peptide bond formation [3,49], 2) positively charged residues [14] or non-optimal codon clusters in the nascent peptide, interacting with ribosome exit tunnel [30,44] or 3) mRNA secondary structure blocking progression of translating ribosomes. [60,23]. However, it is unclear how widespread each of these causes are and to what extent these are functional.…”

Section: Introductionmentioning

confidence: 99%

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Deep conservation of ribosome stall sites across RNA processing genes

Chyżyńska

Labun

Jones

et al. 2020

Preprint

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The rate of translation can vary considerably depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the required time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pause and stall sites, but due to library-specific biases, these predictions are often unreliable.Here, we address this by taking advantage of the deep conservation of the protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important positions of ribosome slowdown - here collectively called stall sites. We analyze multiple ribosome profiling datasets from a phylogenetically diverse group of eukaryotes: yeast, fruit fly, zebrafish, mouse, and human and identify conserved stall sites. We find thousands of stall sites across multiple species, with proline, glycine, and negatively charged amino acids being the main facilitators of stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved regulatory effect on RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

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Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes

Cited by 43 publications

References 82 publications

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Deep conservation of ribosome stall sites across RNA processing genes

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