The total mRNA concentration buffering system in yeast is global rather than gene-specific

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates (SRs) along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentrations and their SRs in non sense-mediated decay (NMD) targets in euploid strains. We found that the altered SRs in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not balanced. In addition, the stability of NMD targets was not specifically counterbalanced by the changes in SR. We conclude that there is no genetic compensation of NMD mRNA targets, and total mRNA buffering uses mostly a global system rather than a gene-specific one in yeast.

PSXIII-33 Polyunsaturated fatty acid and their fetal programming effect on the FFARs from ewe lambs’ ovaries

Free fatty acids (FA) receptors (FFAR) are critical for metabolic functions and energy homeostasis. The FFAR1 and FFAR4 are activated by long-chain polyunsaturated FA, but FFAR2 and FFAR3 join to short-chain FA. There is no evidence fetal programming on nutritional effects on those receptors, insulin receptor (INS-R) and stereological enzyme in lambs’ ovaries. The objective of this study was to evaluate the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation to ewes during late gestation on finishing lamb ovary gene expression. Lambs born from ewes supplemented with Ca salts of EPA and DHA (PUFA), or palm FA distillate (PFAD) high in palmitic and oleic acid at 0.39% DM during the last 50d of gestation were used. After weaning and high concentrate diet adaptation (45d), 14 Hampshire x Dorset cross females lambs were blocked by initial BW and used in a 2x2 factorial arrangement of treatments using the factors of dam supplementation and lamb diets (PUFA or PFAD at 1.5% DM). At day 42, lambs were euthanized and ovary samples were obtained for FFARs, INS-R and steroid acute regulatory protein (STAR) mRNA concentration analysis. There was a dam diet effect on the abundance of FFAR4 mRNA (P = 0.05); lambs born from PUFA dams showed greater concentration than PFAD. Also, PUFA lambs tend to have greater concentration of FFAR4 (P = 0.07). The abundance of FFAR2, INS-R, and STAR mRNA was not significant for dam or lamb diet effect (P > 0.1). The mRNA of FFAR1 and FFAR3 were no found in the samples. However, mRNA concentration of FFAR2 and STAR were positively associated (r = 0.74 P < 0.05). In conclusion, dam and lamb diets modified FFAR4 mRNA concentration; and there might be a functional association between FFAR2 and STAR.

338 Effect of eicosapentaenoic acid and docosahexaenoic acid supplementation on placenta and fetal liver mRNA, and fetal liver and brain fatty acids profile on early gestation in sheep

Polyunsaturated fatty acids supplementation in late gestation change offspring metabolism; however, their effect is not well known on early gestation in ewes. The objectives of this study were to determine the effect of dietary supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in pregnant ewes on the concentration of EPA and DHA on fetal liver (FL) and fetal central nervous system (FCNS), and to evaluate the effect of the supplementation with EPA+ DHA on mRNA expression of genes associated with transport and metabolism of fatty acids (FA) in FL and placenta (caruncles and cotyledons). Twelve ewes (4 pens, three per pen) were blocked by pregnancy day. The ewes were assigned during the first 45 d of gestation to diet with an addition of 1.5% (dry matter bases) monounsaturated FA (MUFA) or EPA+DHA. A C-section was conducted at d 45 of gestation to collect FL, FCNS, caruncle and cotyledon. Data were analyzed using a mixed procedure (SAS). For the placenta mRNA concentration, a 2x2 factorial was used considering caruncle and cotyledon as the second main factor. Isomers of C18:1 (t6,8 and t12) increase (P < 0.05) in FL and FCNS with MUFA supplementation, fatty acids C20:3 (n-6), C20:3 (n-3), C22:1, C22:5 and C22:6 increase (P < 0.05) in FL and FCNS with EPA+DHA supplementation. In FL there was a tendency to increase for mRNA expression of FATP-1 (P = 0.10) with EPA+DHA supplementation, while mRNA concentration for LPL was greater (P = 0.02) for MUFA supplementation. In placenta DNMT3b and FFAR-4 showed a significant FA x tissue interaction (P < 0.05). These results suggest that FA supplementation during early gestation alters the FA profile in FL and FCNS and changed mRNA concentration of genes involved in the transport of FA and cell metabolism.

339 Effect of DHA and EPA supplementation during the first third of gestation on growth, metabolism and gene expression in hypothalamus in finished lambs

Supplementation with fatty acids (FA) during late gestation regulates offspring development; however, its effect in the first third of gestation is unknown. The objective of this study was to evaluate the effects of with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation during the first third of gestation on productive performance and hypothalamic neuropeptides on the offspring. Seventy-nine post-weaning lambs, born of sheep supplemented in the first third of gestation with 1.5% Ca salts rich in monounsaturated FA (MUFA) or PUFA [DHA and EPA; Dam supplementation (DS)], were distributed in a 2x2 factorial arrangement of treatments to finishing diets containing MUFA or PUFA [Lamb supplementation (LS)]. The experiment last for 56 d. Measurements dry matter intake (daily), body weight and plasma metabolites (every 14 d) were taken. Twenty-four lambs were slaughtered and hypothalamus were obtained for mRNA concentration. The data were analyzed with a mixed model in SAS (9.4) using repeated measurements. There was a DS x LS interaction for BW (P < 0.10) where LS with PUFA born from DS with MUFA were heavier than the other 3 treatments. Lambs born from DS with MUFA have a greater DMI (P < 0.01). There was a time x DS effect (P < 0.05) for plasma glucose and non-esterified fatty acids (NEFA) concentration, due to a greater concentration for glucose and lower for NEFA on the lambs born from DS-MUFA at weaning but not on d 56. Lambs born from MUFA supplemented dams had a greater (P ≤ 0.05) hypothalamus mRNA concentration for CART, GH-receptor, KISS-1, leptin receptor, POMC, and NPY receptor Y1. There were no LS effects (P > 0.05) for these neuropeptides. These results indicate that supplementation with fatty acids during early pregnancy change productive performance, metabolic, and neuropeptides of lambs independently of the final diet.

ETFL: A formulation for flux balance models accounting for expression, thermodynamics, and resource allocation constraints

Since the introduction of metabolic models and flux balance analysis (FBA) in systems biology, several attempts have been made to add expression data. However, directly accounting for enzyme and mRNA production in the mathematical programming formulation is challenging because of macromolecules, which introduces a bilinear term in the mass-balance equations that become harder to solve than linear formulations like FBA. Furthermore, there have been no attempts to include thermodynamic constraints in these formulations, which would yield an even more complex mixed-integer non-linear problem.We propose here a new framework, called Expression and Thermodynamics Flux (ETFL), as a new ME-model implementation. ETFL is a top-down model formulation, from metabolism to RNA synthesis, that simulates thermodynamic-compliant intracellular fluxes as well as enzyme and mRNA concentration levels. The formulation results in a mixed-integer linear problem (MILP) that enables both relative and absolute metabolite, protein, and mRNA concentration integration. The proposed formulation is compatible with mainstream MILP solvers and does not require a non-linear solver. It also accounts for growth-dependent parameters, such as relative protein or mRNA content.We present here the formulation of ETFL along with its validation using results obtained from a well-characterizedE. colimodel. We show that ETFL is able to reproduce proteome-limited growth, which FBA cannot. We also subject it to different analyses, including the prediction of feasible mRNA and enzyme concentrations in the cell, and propose ETFL-based adaptations of other common FBA-based procedures.The software is available on our public repository athttps://github.com/EPFL-LCSB/etfl.Author summaryMetabolic modeling is a useful tool for biochemists who want to tweak biological networks for the direct expression of key products, such as biofuels, specialty chemicals, or drug candidates. To provide more accurate models, several attempts have been made to account for protein expression and growth-dependent parameters, key components of biological networks, though this is computationally challenging, especially when also attempting to include thermodynamics. To the best of our knowledge, there is no published methods integrating these three types of constraints in one model. We propose here a transparent mathematical formulation to model both expression and metabolism of a cell, along with a reformulation that allows a computationally tractable inclusion of growth-dependent parameters and thermodynamics. We demonstrate good performance using community-standard software, and propose ways to adapt classical modeling studies to expression-enabled models. The incorporation of thermodynamics and growth-dependent variables provide a finer modeling of expression because they eliminate thermodynamically unfeasible solutions and consider phenotypic differences in different growth regimens, which are key for accurate modeling.

Quantitative study of the somitogenetic wavefront in zebrafish

ABSTRACTA quantitative description of the molecular networks that sustain morphogenesis is one of the challenges of developmental biology. Specifically, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. Here we quantitatively study and perturb the network in zebrafish that sustains this wavefront and compare our observations to a model whereby the wavefront is due to a switch between stable states resulting from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of ERK and of ERK on RA synthesis. This model quantitatively accounts for the near linear shortening of the post-somitic mesoderm (PSM) in response to the observed exponential decrease during somitogenesis of the mRNA concentration of a morphogen (Fgf8). It also accounts for the observed dynamics of the PSM when the molecular components of the network are perturbed. The generality of our model and its robustness allows for its test in other model organisms.

Robotic Platform for the Delivery of Gene Products Into Single Cells in Organotypic Slices of the Developing Mouse Brain

Microinjection of genetic components and dye into organotypic slices provides excellent single cell resolution for unraveling biological complexities, but is extremely difficult and time consuming to perform manually resulting in low yield and low use in the developmental biology field. We developed a computer vision guided platform to inject specimen with mRNA, and/or dye and investigated the efficiency of the process using organotypic slices of the mouse developing neocortex. We demonstrate that the system significantly increases yield of injection relative to manual use by an order of magnitude, allows for cell tracking over 0, 24, and 48 hours post injection in culture, and enables mRNA translation of injected product. The autoinjector platform thus can open the door to new types of experiments including investigating effects of mRNA concentration, and composition on cell fate, and tracking these effects on cell reprogramming and lineage.