Characterization of the Export of Bulk Poly(A)+ mRNA in Saccharomyces cerevisiae during the Wine-Making Process

Ethanol stress affects the nuclear export of mRNA similarly to heat shock in Saccharomyces cerevisiae. However, we have little information about mRNA transport in actual alcoholic fermentation. Here we characterized the transport of mRNA during wine making and found that bulk poly(A)⁺ mRNA accumulated in the nucleus as fermentation progressed.     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

An agent-based model for mRNA export through the nuclear pore complex

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.     

Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone. The degree of expression of the mammalian System A activity in Xenopus oocytes corresponded to the known transport rates in the tissue from which the mRNA was prepared. For example, hepatic mRNA from glucagon-treated rats produced greater System A activity than mRNA from control animals, and the mRNA from the CHO transport mutant cell line alar4-H3.9, which overproduces System A, resulted in higher transport rates than mRNA from the parental cell line (CHO-K1). Fractionation of total mRNA poly(A+) by nondenaturing agarose gel electrophoresis revealed transport activity associated with a 2.0-2.5-kilobase mRNA fraction common to each of the three tissues tested.     

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.     

In Vivo Colocalisation of oskar mRNA and Trans-Acting Proteins Revealed by Quantitative Imaging of the Drosophila Oocyte

Efficient mRNA transport in eukaryotes requires highly orchestrated relationships between nuclear and cytoplasmic proteins. For oskar mRNA, the Drosophila posterior determinant, these spatio-temporal requirements remain opaque during its multi-step transport process. By in vivo covisualization of oskar mRNA with Staufen, its putative trafficking protein, we find oskar mRNA to be present in particles distinct from Staufen for part of its transport. oskar mRNA stably associated with Staufen near the posterior pole. We observe oskar mRNA to oligomerize as hundreds of copies forming large particles which are necessary for its long range transport and localization. We show the formation of these particles occurs in the nurse cell nucleus in an Hrp48-dependent manner. We present a more refined model of oskar mRNA transport in the Drosophila oocyte.     

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.     

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.     

Involvement of the late secretory pathway in actin regulation and mRNA transport in yeast

Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization.     

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.     

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.     

A Comparative Quantitative Assessment of Axonal and Dendritic mRNA Transport in Maturing Hippocampal Neurons

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.     

Relationship between sugar uptake kinetics and total sugar consumption in different industrialSaccharomyces cerevisiae strains during alcoholic fermentation

Summary The sugar transport, fermentative alcohol dehydrogenase (ADH) and protease activities of different industrial strains ofSaccharomyces cerevisiae were measured during batch alcoholic fermentation.These strains exhibited different apparent loss of activity of sugar transport, which seemed to be characteristic of each one. A good correlation was found systematically between the integration of sugar transport activity along fermentation and the maximum amount of sugar consumed during fermentation. In all strains sugar transport activity exhibit a lower half-time than fermentative ADH activity. These progressive declines of both sugar uptake and ADH activities ofSaccharomyces cerevisiae during batch fermentation seemed to not result from an increase in the protease activity.     

A complex peptide-sorting signal, but no mRNA signal, is required for the Sec-independent transport of Ist2 from the yeast ER to the plasma membrane

The yeast integral plasma membrane protein Ist2 belongs to a group of membrane proteins which are synthesized from localized mRNAs. The protein reaches the plasma membrane via the ER on a route operating independently of the classical secretory pathway. We have identified a complex peptide-sorting signal located at the extreme C-terminus. This sorting signal operates independently of targeting information in IST2 mRNA and sorting to the plasma membrane does not require She-mediated mRNA transport into daughter cells. Based on these results, we suggest a posttranslational mechanism, which leads to the concentration of Ist2--via multimerization--at ER sites, followed by direct transport to the plasma membrane. This novel mechanism operates downstream of IST2 mRNA localization.     

Induction of glycinebetaine uptake into Xenopus oocytes by injection of poly(A)+ RNA from renal cells exposed to high extracellular NaCl

Madin-Darby canine kidney (MDCK) cells accumulate glycinebetaine via Na(+)-dependent transport in response to hypertonic stress. When extracellular tonicity is increased by the addition of NaCl, Vmax for glycinebetaine transport increases without an associated change in Km, consistent with an increase in the number of functioning transporters. To test whether increased transport activity results from increased gene expression, we injected poly(A)+ RNA (mRNA) from MDCK cells into Xenopus oocytes and assayed for glycinebetaine uptake in ovo. RNA-induced Na(+)-dependent uptake is observed in oocytes injected with mRNA from cells exposed to high extracellular NaCl, but not in oocytes injected with either water or mRNA from cells maintained in isotonic medium. Unfractionated mRNA induces glycinebetaine uptake in ovo at a rate which is approximately 3-fold higher than in water-injected controls. Size-fractionated mRNA (median size 2.8 kilobases) induces uptake at a rate which is approximately 7-fold higher than controls. Such RNA-induced transport activity in ovo is consistent with heterologous expression of Na(+)/glucinebetaine cotransporters encoded by renal mRNA. Increased transporter mRNA in cells exposed to hypertonicity probably underlies the pattern of expression observed in ovo. This can account for the observed rise in MDCK cell glycinebetaine transport during hypertonic stress.     

Remarks on the metabolism of kluyveromyces lactis van der walt

Kluyveromyces lactis CBS 683, showed no Crabtree effect but the Pasteur effect was very strong. It could be seen that the metabolism of glucose was adaptative. The anaerobic fermentation of lactose was always greater than the anaerobic fermentation on glucose and galactose. The transport across wall and cell membrane of glucose and galactose limits the fermentation rate of these substrates.