Translatomics: The Global View of Translation

Zhao, Jing; Qin, Bo; Nikolay, Rainer; Spahn, C.M.T.; Zhang, Gong

doi:10.3390/ijms20010212

Cited by 70 publications

(67 citation statements)

References 145 publications

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“…The wide-spread utilisation of polysome profiling has nevertheless been historically hindered by its intrinsic complexity, time-consuming nature, limited capacity for high throughput adaptation and its requirement for relatively large initial sample sizes [27]. The most commonly applied protocols utilise sucrose gradients, of varying concentrations, with volumes of~12 mL, most frequently in the Beckman SW41Ti rotor [7,9,13,28], and the technique has been successfully scaled up using SW28Ti rotor tubes (with 38 mL gradients) to accommodate up to 45 OD 260 nm units of cell extract [10,29].…”

Section: Introductionmentioning

confidence: 99%

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method

Mašek

Llano

Gahurová

et al. 2020

IJMS

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Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)—a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

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Section: Introductionmentioning

confidence: 99%

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method

Mašek

Llano

Gahurová

et al. 2020

IJMS

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“…Emerging evidence suggests that regulatory control at the level of mRNA translation contributes significantly to gene expression and function (1)(2)(3)(4). Consistent with this evidence, recent findings indicate that coordinated changes in post-transcriptional regulatory networks can alter cellular phenotype and behavior (2,5).…”

Section: Introductionmentioning

confidence: 68%

CELF1 is an EIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

Chaudhury

Pal

Kongchan

et al. 2019

Preprint

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Mounting evidence is revealing a granularity within gene regulation that occurs at the level of mRNA translation. Within mammalian cells, canonical cap-dependent mRNA translation is dependent upon the interaction between the m 7 G cap-binding protein eukaryotic initiation factor 4E (eIF4E) and the scaffolding protein eukaryotic initiation factor 4G (eIF4G), the latter of which facilitates pre-translation initiation complex assembly, mRNA circularization, and ultimately ribosomal scanning. In breast epithelial cells, we previously demonstrated that the CELF1 RNA-binding protein promotes the translation of epithelial to mesenchymal transition (EMT) effector mRNAs containing GU-rich elements (GREs) within their 3' untranslated regions (UTRs). Here we show that within this context, CELF1 directly binds to both the eIF4E capbinding protein and Poly(A) binding protein (PABP), promoting translation of GRE-containing mRNAs in mesenchymal cells. Disruption of this CELF1/eIF4E interaction inhibits both EMT induction and experimental metastasis. Our findings illustrate a novel way in which noncanonical mechanisms of translation initiation underlie transitional cellular states within the context of development or human disease.It has now been well-documented that both oncogenes and signaling pathways regulating expression of these oncogenes together converge on the regulation of mRNA translation (2,15).Indeed, within the process of cellular transformation, mechanisms at each stage of translation and the ribosomal machinery may be co-opted to fostering translation that perpetuates the oncogenic program (16)(17)(18). This motivated us to more closely examine how CELF1 regulates translation of GRE-containing EMT effector mRNAs during EMT of breast epithelial cells. Results CELF1 interacts directly with eIF4E and PABP at the m 7 G cap, independent of intact eIF4G1Our previous work suggested that CELF1's control of GU-rich element-containing EMT effector mRNA translation is mediated at the level of 5' m 7 G cap-dependent translation initiation (10). We first confirmed and extended these findings by examining an additional CELF1 regulatory target within this context. We fused the 3' UTRs of a subset of GRE-containing mRNAs (JUNB, CRLF1, SNAI1, and SSBP2) downstream of the Renilla luciferase coding sequence in the pRL-TK-CXCR4-6x (19) reporter plasmid (Figure 1a). Transfection of the GREcontaining 3' UTR reporters into MCF10A cells, either untreated or treated with TGF-β to induce EMT, revealed a significant increase in reporter activity specific to treated cells Figure 1a), independent of any differences in relative mRNA expression (Figure 1b). In parallel, we built a battery of bicistronic constructs in which the same thymidine kinase promoter was utilized to drive expression of the firefly luciferase coding sequence, followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), the Renilla luciferase open reading frame and individual GRE-containing 3' UTRs (pFR-EMCV) (Figure 1c). In contrast to cap-depen...

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“…One particular application of the translation models consists in deciphering the results of application of modern sequencing-based technologies quantifying the global state of the translational machinery, such as Ribo-Seq or TRAP-Seq [33]. The amount of data of this type (translatomic data) rapidly grows, but remains less ready for intuitive interpretation compared to other omics data types and require mathematical modeling, taking into account various aspects of polysome [34].…”

Section: Discussionmentioning

confidence: 99%

Basic, simple and extendable kinetic model of protein synthesis

Gorban

Harel‐Bellan

Morozova

et al. 2019

Mathematical Biosciences and Engineering

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Protein synthesis is one of the most fundamental biological processes, which consumes a significant amount of cellular resources. Despite existence of multiple mathematical models of translation, varying in the level of mechanistical details, surprisingly, there is no basic and simple chemical kinetic model of this process, derived directly from the detailed kinetic model. One of the reasons for this is that the translation process is characterized by indefinite number of states, thanks to existence of polysomes. We bypass this difficulty by applying a trick consisting in lumping multiple states of translated mRNA into few dynamical variables and by introducing a variable describing the pool of translating ribosomes. The simplest model can be solved analytically under some assumptions. The basic and simple model can be extended, if necessary, to take into account various phenomena such as the interaction between translating ribosomes, limited amount of ribosomal units or regulation of translation by microRNA. The model can be used as a building block (translation module) for more complex models of cellular processes. We demonstrate the utility of the model in two examples. First, we determine the critical parameters of the single protein synthesis for the case when the ribosomal units are abundant. Second, we demonstrate intrinsic bi-stability in the dynamics of the ribosomal protein turnover and predict that a minimal number of ribosomes should pre-exists in a living cell to sustain its protein synthesis machinery, even in the absence of proliferation.

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Translatomics: The Global View of Translation

Cited by 70 publications

References 145 publications

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method

CELF1 is an EIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

Basic, simple and extendable kinetic model of protein synthesis

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