scholarly journals Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

mSystems ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Jan Karlsen ◽  
Johannes Asplund-Samuelsson ◽  
Quentin Thomas ◽  
Michael Jahn ◽  
Elton P. Hudson
Keyword(s):  
Gene Regulation ◽  
Stress Response ◽  
Translational Control ◽  
Carbon Starvation ◽  
Ribosome Profiling ◽  
Untranslated Regions ◽  
Translational Efficiency ◽  
Data Set ◽  
Stress Response Genes ◽  
Regulation Strategies

ABSTRACT 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.

scholarly journals Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

2015 ◽  
Author(s):  
David E Weinberg ◽  
Premal Shah ◽  
Stephen W Eichhorn ◽  
Jeffrey A Hussmann ◽  
Joshua B Plotkin ◽  
...  
Keyword(s):  
Translational Control ◽  
Nucleotide Composition ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Coding Regions ◽  
Genome Wide ◽  
Upstream Open Reading Frames ◽  
Improved Methods ◽  
Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

Specialized Ribosomes: A New Frontier in Gene Regulation, Organismal Biology, & Evolution

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-41-SCI-41
Author(s):  
Maria Barna
Keyword(s):  
Gene Regulation ◽  
Translational Control ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Mammalian Genome ◽  
Ribosome Profiling ◽  
Human Diseases ◽  
Biological Information ◽  
Control Of Gene Expression

Abstract The central dogma of molecular biology has for decades served as an explanation for the flow of genetic information within a biological system. In so far as the normal flow of biological information from mRNA to protein, the ribosome has been perceived to decode the genome with essentially machine-like precision; serving as an integral but largely passive participant in the synthesis of all effector proteins across all kingdoms of life. Importantly, a large class of human diseases collectively known as 'ribosomopathies' are characterized by mutations in ribosome components that lead to devastating human conditions including bone marrow failure for which the underlying molecular basis remains poorly understood. In this respect, our research has changed the view that ribosomes carry out largely rote-like functions by demonstrating that not all of the millions of ribosomes within each cell are the same and that ribosome heterogeneity provides a novel means for diversity of the proteins that can be produced in specific cells, tissues, and organisms. I will present our work centered on providing a roadmap for the characterization of ribosome composition at a single cell level and during cellular differentiation. We employed a highly quantitative mass spectrometry-based approach to precisely quantify the abundance of each ribosomal protein (RP) as well as a large cohort of auxiliary ribosome associating factors belonging to actively translating ribosomes within embryonic stem cells. This led to the identification of a subset of ribosomes that are heterogeneous for RP composition. To further address the functional role of ribosome heterogeneity in translational control of the mammalian genome, we employed CRISPR/Cas9 to endogenously tag and purify heterogeneous ribosome populations. We then developed an adapted ribosome profiling method to precisely quantify and characterize the nature of mRNAs translated by distinct heterogenous ribosomes genome-wide. This led to the identification of subpools of transcripts, critical for key cellular processes including cell signaling, metabolism, growth, proliferation and survival, which are selectively translated by specific types of ribosomes. Most remarkably, there are specific metabolic pathways where almost every single component is selectively translated by specialized ribosomes demarcated by a single RP. I will further present recent findings on the mechanisms by which ribosome-mediated control of gene expression is encoded by structured RNA regulons within 5'UTRs. Together, these studies reveal a critical link between ribosome heterogeneity and specialized translational control of the mammalian genome, which adds an important layer of control to the post-transcriptional circuitry of gene regulation and may be critically perturbed in human diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dynamic translation regulation inCaulobactercell cycle control

2016 ◽  
Vol 113 (44) ◽  
pp. E6859-E6867 ◽  
Author(s):  
Jared M. Schrader ◽  
Gene-Wei Li ◽  
W. Seth Childers ◽  
Adam M. Perez ◽  
Jonathan S. Weissman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Transcriptional Control ◽  
Relative Rate ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Translational Efficiency ◽  
Specific Cell ◽  
Control Of Gene Expression ◽  
Regulatory Pathways

Progression of theCaulobactercell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from—but highly coordinated with—the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of theCaulobactercell cycle.

scholarly journals Ribosome profiling elucidates the contribution of differential translation to differential gene expression in bundle sheath and mesophyll cells in maize

2020 ◽  
Author(s):  
Prakitchai Chotewutmontri ◽  
Alice Barkan
Keyword(s):  
Gene Expression ◽  
Differential Gene Expression ◽  
Translational Control ◽  
C4 Photosynthesis ◽  
Bundle Sheath ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
M Cells ◽  
Rna Seq ◽  
Differential Gene

The efficiencies offered by C4 photosynthesis have motivated efforts to understand its biochemical, genetic and developmental basis. Reactions underlying C4 traits in most C4 plants are partitioned between two cell types, bundle sheath (BS) and mesophyll (M) cells. RNA-seq has been used to catalog differential gene expression in BS and M cells in maize and several other C4 species. However, the contribution of translational control to maintaining the distinct proteomes of BS and M cells has not been addressed. In this study, we used ribosome profiling (ribo-seq) and RNA-seq to describe translatomes, translational efficiencies, and microRNA abundance in BS and M-enriched fractions of maize seedling leaves. A conservative interpretation of our data revealed 39 genes exhibiting cell-type dependent differences in translational efficiency, fourteen of which encode proteins with core roles in C4 photosynthesis. Use of more relaxed criteria that are widely used in ribo-seq studies increased the set of differentially translated genes to several hundred. In addition, our data expand the set of genes known to be differentially expressed in BS and M cells, including genes encoding transcription factors and microRNAs. These data add to the resources for understanding the evolutionary and developmental basis of C4 photosynthesis and for its engineering into C3 crops.

scholarly journals Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast

2020 ◽  
Author(s):  
Angela Rubio ◽  
Sanjay Ghosh ◽  
Michael Mülleder ◽  
Markus Ralser ◽  
Juan Mata
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Fission Yeast ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
Mapk Pathway ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Ribosome Stalling ◽  
Response To Stress

Abstract Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.

scholarly journals AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yanxing Ma ◽  
Hainan Tian ◽  
Rao Lin ◽  
Wei Wang ◽  
Na Zhang ◽  
...  
Keyword(s):  
Stress Response ◽  
Protein Phosphatase ◽  
Aba Signaling ◽  
Response Gene ◽  
Stress Response Genes ◽  
Arabidopsis Protein ◽  
Evolutionarily Conserved ◽  
Increased Sensitivity ◽  
Aba Response ◽  
Transcription Repressors

AbstractExpression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes.

scholarly journals Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

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