Ribosome Profiling Uncovers the Role of uORFs in Translational Control of Gene Expression during Erythroblast Differentiation

Abstract The erythroid progenitor compartment possesses a large expansion capacity, both in vivo and in vitro, which enables a rapid restoration of peripheral erythrocytes following severe blood loss. This expansion is tightly regulated to maintain erythrocyte numbers between narrow boundaries, and to balance expansion of the erythroid compartment against the availability of iron for heme and haemoglobin production. We previously observed that control of mRNA translation is crucial for expansion of the erythroid compartment. We also showed that translation of specific transcripts is impaired in Diamond Blackfan Anemia (DBA), a severe congenital anemia due to defective ribosome biosynthesis. Transcripts can be subject to translational control through domains in the 5’- or 3’UTR, including secondary structures, protein binding sequences and upstream open reading frames (uORFs). The presence of uORFs, including those starting at non-AUG codons in the 5’UTR, may alter the level of mRNA translation, but may also result in the expression of alternative protein isoforms because translation initiation may be redirected to more downstream start codons. The aim of our current studies is to provide a genome wide map of mRNA translation efficiency during erythropoiesis that can be used to investigate defective mRNA translation in, for instance, DBA. Ribosome profiling is a genome wide high-throughput sequencing technology for global mapping of translation initiation sites that allows translation analysis with codon resolution at the genome wide level. We first investigated translational changes occurring during differentiation of mouse erythroblasts. We used p53-deficient, growth factor dependent and differentiation competent immortalized erythroblast cultures that were expanded in presence of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids as T0, and subsequently differentiated the cells in presence of Epo for 17 and 46 hours (T17, and T46 samples). To obtain ribosome footprints, the cells were treated for 7 minutes with harringtonin or solvent, and subsequently for 5 minutes with cycloheximide, which arrests translation by stabilizing the ribosomes at translation initiation codons, or on all codons, respectively. We used optimized protocols for ribosome footprinting and data analysis, and focused the analysis on transcripts containing uORFs. First we performed a qualitative analysis of start codon usage. The ribosome footprint data proved to be superior to previously used polyribosome recruitment. In some cases polysome recruitment appeared to represent translation of an uORFs while the protein coding ORF is hardly translated (e.g. Csf2rb2, Puma). In another set of transcripts, we found uORFs that are differentially translated during differentiation, and thereby regulate differential translation from a downstream start codon (e.g. Klf3, Use1, CD47, Kell). Finally, comparison of ribosome footprints determined in erythroblasts and in myoblasts/myotubes revealed tissue specific translation regulation of otherwise ubiquitously expressed transcripts among which transcripts encoding ribosomal proteins. Second, we will perform quantitative analysis of mRNA translation in erythropoiesis through the comparison of ribosome footprint reads in an ORF with total mRNA reads obtained from total mRNA sequencing of the same sample. The obtained insight in transcript specific translation at codon resolution is of great value to understand many cellular processes during erythropoiesis, and will be of particular interest to understand responses to iron availability and reactive oxygen species that particularly affect translation of transcripts harboring uORFs. Disclosures No relevant conflicts of interest to declare.

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Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors

10.1101/216234 ◽

2017 ◽

Author(s):

Nahuel A. Paolini ◽

Kat S. Moore ◽

Franca M. di Summa ◽

Ivo F.A.C. Fokkema ◽

Peter A.C. ‘t Hoen ◽

...

Keyword(s):

Translation Initiation ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translation Initiation Factor ◽

List Type ◽

Translation Efficiency ◽

Proteotoxic Stress ◽

Advantages And Disadvantages ◽

Ribosome Density ◽

Eif2 Phosphorylation

AbstractThe regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how eIF2 phosphorylation affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict eIF2-dependent translation efficiency, but uORF identity differs. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.- eif2 dependent translation in erythroblasts during proteotoxic stress determined by ribosome footprinting- identification of transcription factors upregulated in response to eIF2 phosphorylation- Advantages and disadvantages of translation initiation site determination using harringtonine- distinct uORF pattern in transcripts with enhanced, or more than average reduced translation upon proteotoxic stress

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Defining the anti-Shine-Dalgarno sequence interaction and quantifying its functional role in regulating translation efficiency

10.1101/038984 ◽

2016 ◽

Author(s):

Adam J Hockenberry ◽

Adam R Pah ◽

Michael C Jewett ◽

Luís AN Amaral

Keyword(s):

Functional Role ◽

Functional Form ◽

Ribosome Profiling ◽

Start Codon ◽

Translation Efficiency ◽

Gfp Expression ◽

Genome Wide ◽

Shine Dalgarno Sequence ◽

Genome Scale ◽

The Relationship

Studies dating back to the 1970s established that binding between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes and mRNA helps to facilitate translation initiation. The location of aSD binding relative to the start codon, the full extents of the aSD sequence, and the functional form of the relationship between aSD binding and translation efficiency are important parameters that remain ill defined in the literature. Here, we leverage genome-wide estimates of translation efficiency to determine these parameters and show that anti-Shine-Dalgarno sequence binding increases the translation of endogenous mRNAs on the order of 50%. Our findings highlight the non-linearity of this relationship, showing that translation efficiency is maximized for sequences with intermediate aSD binding strengths. These mechanistic insights are highly robust; we find nearly identical results in ribosome profiling datasets from 3 highly diverged bacteria, as well as independent genome-scale estimates and controlled experimental data using recombinant GFP expression.

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Computational and Experimental Tools to Monitor the Changes in Translation Efficiency of Plant mRNA on a Genome-Wide Scale: Advantages, Limitations, and Solutions

International Journal of Molecular Sciences ◽

10.3390/ijms20010033 ◽

2018 ◽

Vol 20 (1) ◽

pp. 33 ◽

Cited By ~ 5

Author(s):

Irina Goldenkova-Pavlova ◽

Olga Pavlenko ◽

Orkhan Mustafaev ◽

Igor Deyneko ◽

Ksenya Kabardaeva ◽

...

Keyword(s):

Translational Control ◽

Gene Expression Regulation ◽

Plant Genome ◽

Computational Algorithms ◽

Translation Efficiency ◽

Translation Control ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Experimental Approaches

The control of translation in the course of gene expression regulation plays a crucial role in plants’ cellular events and, particularly, in responses to environmental factors. The paradox of the great variance between levels of mRNAs and their protein products in eukaryotic cells, including plants, requires thorough investigation of the regulatory mechanisms of translation. A wide and amazingly complex network of mechanisms decoding the plant genome into proteome challenges researchers to design new methods for genome-wide analysis of translational control, develop computational algorithms detecting regulatory mRNA contexts, and to establish rules underlying differential translation. The aims of this review are to (i) describe the experimental approaches for investigation of differential translation in plants on a genome-wide scale; (ii) summarize the current data on computational algorithms for detection of specific structure–function features and key determinants in plant mRNAs and their correlation with translation efficiency; (iii) highlight the methods for experimental verification of existed and theoretically predicted features within plant mRNAs important for their differential translation; and finally (iv) to discuss the perspectives of discovering the specific structural features of plant mRNA that mediate differential translation control by the combination of computational and experimental approaches.

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Translation efficiency is maintained at elevated temperature inE. coli

10.1101/109264 ◽

2017 ◽

Author(s):

Gareth J Morgan ◽

David H Burkhardt ◽

Jeffery W Kelly ◽

Evan T Powers

Keyword(s):

Heat Shock ◽

Mrna Translation ◽

Cellular Protein ◽

Ribosome Profiling ◽

Melting Transition ◽

Temperature Sensitive ◽

Translation Efficiency ◽

Protein Levels ◽

Mrna Structure ◽

Genome Wide

ABSTRACTCellular protein levels are dictated by the balance between gene transcription, mRNA translation and protein degradation, among other factors. Cells must manage their proteomes during stress; one way in which they may do so, in principle, is by differential translation. We used ribosome profiling to directly monitor translation inE. coliat 30 °C and investigate how this changes after 10-20 minutes of heat shock at 42 °C. Translation is controlled by the interplay of several RNA hybridization processes, which are expected to be temperature sensitive. However, translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat shock response transcriptional program at 30 °C. Several gene-specific parameters correlated with translation efficiency, including predicted mRNA structure and whether a gene is cotranslationally translocated into the inner membrane. Genome-wide predictions of the temperature dependence of mRNA structure suggest that relatively few genes show a melting transition between 30 °C and 42 °C, consistent with our observations. A linear model with five parameters can predict 33% of the variation in translation efficiency between genes, which may be useful in interpreting transcriptome data.

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A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Scientific Reports ◽

10.1038/s41598-021-82025-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Brandon M. Trainor ◽

Arnab Ghosh ◽

Dimitri G. Pestov ◽

Christopher U. T. Hellen ◽

Natalia Shcherbik

Keyword(s):

Gene Expression ◽

Translation Initiation ◽

Translational Control ◽

Rna Binding ◽

Mutational Analysis ◽

Fine Tuning ◽

Translation Efficiency ◽

Control Of Gene Expression ◽

Enhancer Element ◽

Independent Translation

AbstractCap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.

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The Role of Translation Initiation Regulation in Haematopoiesis

Comparative and Functional Genomics ◽

10.1155/2012/576540 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Godfrey Grech ◽

Marieke von Lindern

Keyword(s):

Gene Expression ◽

Translation Initiation ◽

Translational Control ◽

Molecular Mechanisms ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Translation Control ◽

Haematological Disorders

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

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

10.1101/021501 ◽

2015 ◽

Cited By ~ 2

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.

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Genome-wide imaging screen uncovers molecular determinants of arsenite-induced protein aggregation and toxicity

Journal of Cell Science ◽

10.1242/jcs.258338 ◽

2021 ◽

Author(s):

Stefanie Andersson ◽

Antonia Romero ◽

Joana Isabel Rodrigues ◽

Sansan Hua ◽

Xinxin Hao ◽

...

Keyword(s):

Protein Aggregation ◽

Translational Control ◽

Protein Biosynthesis ◽

Cellular Systems ◽

Genome Wide ◽

A Genome ◽

Toxic Metalloid ◽

Arsenic Stress

The toxic metalloid arsenic causes widespread misfolding and aggregation of cellular proteins. How these protein aggregates are formed in vivo, the mechanisms by which they affect cells, and how cells prevent their accumulation is not fully understood. To find components involved in these processes, we performed a genome-wide imaging screen and identified yeast deletion mutants with either enhanced or reduced protein aggregation levels during arsenite exposure. We show that many of the identified factors are crucial to safeguard protein homeostasis (proteostasis) and to protect cells against arsenite toxicity. The hits were enriched for various functions including protein biosynthesis and transcription, and dedicated follow-up experiments highlight the importance of accurate transcriptional and translational control for mitigating protein aggregation and toxicity during arsenite stress. Some of the hits are associated with pathological conditions, suggesting that arsenite-induced protein aggregation may affect disease processes. The broad network of cellular systems that impinge on proteostasis during arsenic stress identified in this current study provides a valuable resource and a framework for further elucidation of the mechanistic details of metalloid toxicity and pathogenesis.

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sPepFinder expedites genome-wide identification of small proteins in bacteria

10.1101/2020.05.05.079178 ◽

2020 ◽

Author(s):

Lei Li ◽

Yanjie Chao

Keyword(s):

De Novo ◽

Bacterial Species ◽

Computational Prediction ◽

Ribosome Profiling ◽

Support Vector ◽

Initiation Rate ◽

E Coli ◽

Small Proteins ◽

Genome Wide ◽

A Genome

ABSTRACTSmall proteins shorter than 50 amino acids have been long overlooked. A number of small proteins have been identified in several model bacteria using experimental approaches and assigned important functions in diverse cellular processes. The recent development of ribosome profiling technologies has allowed a genome-wide identification of small proteins and small ORFs (smORFs), but our incomplete understanding of small proteins hinders de novo computational prediction of smORFs in non-model bacterial species. Here, we have identified several sequence features for smORFs by a systematic analysis of all the known small proteins in E. coli, among which the translation initiation rate is the strongest determinant. By integrating these features into a support vector machine learning model, we have developed a novel sPepFinder algorithm that can predict conserved smORFs in bacterial genomes with a high accuracy of 92.8%. De novo prediction in E. coli has revealed several novel smORFs with evidence of translation supported by ribosome profiling. Further application of sPepFinder in 549 bacterial species has led to the identification of > 100,000 novel smORFs, many of which are conserved at the amino acid and nucleotide levels under purifying selection. Overall, we have established sPepFinder as a valuable tool to identify novel smORFs in both model and non-model bacterial organisms, and provided a large resource of small proteins for functional characterizations.

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Seeking a Role for Translational Control by Alternative Polyadenylation in Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms9091885 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1885

Author(s):

Rachael E. Turner ◽

Traude H. Beilharz

Keyword(s):

Saccharomyces Cerevisiae ◽

Translational Control ◽

Model Organism ◽

Alternative Polyadenylation ◽

Ribosome Profiling ◽

Translation Efficiency ◽

Mrna Isoforms ◽

Cleavage And Polyadenylation ◽

Made In

Alternative polyadenylation (APA) represents an important mechanism for regulating isoform-specific translation efficiency, stability, and localisation. Though some progress has been made in understanding its consequences in metazoans, the role of APA in the model organism Saccharomyces cerevisiae remains a relative mystery because, despite abundant studies on the translational state of mRNA, none differentiate mRNA isoforms’ alternative 3′-end. This review discusses the implications of alternative polyadenylation in S. cerevisiae using other organisms to draw inferences. Given the foundational role that research in this yeast has played in the discovery of the mechanisms of cleavage and polyadenylation and in the drivers of APA, it is surprising that such an inference is required. However, because advances in ribosome profiling are insensitive to APA, how it impacts translation is still unclear. To bridge the gap between widespread observed APA and the discovery of any functional consequence, we also provide a review of the experimental techniques used to uncover the functional importance of 3′ UTR isoforms on translation.

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