Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation

Cordycepin (3′ deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3′ hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3′ end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3′ end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor

Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood. Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro. Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs. These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition. Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general. RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF. Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo. A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3′-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3′-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3′-end cleavage and polyadenylation, that is, cotranscriptionally.

Comparative study on performance of yeast and bacterial membrane bioreactors for high salinity wastewater treatment

Two laboratory-scale membrane bioreactor systems were investigated to treat high saline wastewater containing 1,000 mg/L COD and 32 g/L NaCl, namely: the yeast membrane bioreactor (YMBR) and the bacterial membrane bioreactor (BMBR). COD removal of both processes was above 90% at a hydraulic retention time (HRT) of 5 hours (volumetric loading of 5 kg COD/m³.d), sludge retention time (SRT) of 50 days (the MLSS of above 14 g/L and the F/M of 0.4 d-1). Under these operating conditions, the YMBR could run at a ten-fold lower transmembrane pressure with significantly reduced membrane fouling rate compared to BMBR. This may be because of low production of adhesive extracellular polymers (ECP) and the secondary filtration layer formed from large yeast cells. ECP production of bacterial sludge was increased considerably at high salt concentrations (32 g/L and 45 g/L) and long SRTs. For the bacterial sludge, the increased salinity led to increase in ECP, whereas the ECP content of the yeast sludge was relatively small.

Study on the fouling phenomena of poly(vinylidene fluoride) (PVDF) membrane by protein and yeast

This sub-collection is the result of an investigation into the mechanism of organic fouling in membrane filtration processes. In this experiment, poly(vinylidene fluoride) (PVDF) membranes were used to filter two types of organic foulants, protein and yeast with a concentration of 50mg/l and 20 mg/l, respectively, from suspension in a dead-end filtration cell. These model foulants were stained with fluorescent dyes before filtration. This dataset contains a stack of images of the fouling layer on the PVDF membrane surface captured by a confocal laser scanning microscope (CLSM) and its associated acquisition software. This dataset would be useful to researchers who are investigating the membrane organic fouling mechanism so that new membrane materials and new anti-fouling surface treatment technologies can be developed for water and wastewater industry in the future.

Evaluating backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by protein and yeast

This collection is the result of an investigation into the backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by two types of organic foulants, protein and yeast. In this experiement, poly(vinylidene fluoride) (PVDF) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PVDF membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of poly(vinylidene fluoride) (PVDF) membranes' fouling layer when two types of organic foulants (protein and yeast) present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers who are evaluating the backwashing efficiency of PVDF membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water.

Evaluating backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by yeast and sodium alginate

This collection is the result of an investigation into the backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by yeast and sodium alginate. In this experiement, poly(vinylidene fluoride) (PVDF) membrane was used to filter two types of organic foulants from suspensions in a dead-end stirred cell. The organic foulants including yeast and sodium alginate were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PVDF membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. The data collection contains image data of poly(vinylidene fluoride) (PVDF) membranes' fouling layer when two types of organic foulants (yeast and sodium alginate) are present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. The collection would be useful to researchers evaluating the backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water.

Evaluating backwashing efficiency of polycarbonate (PC) membrane fouled by protein, sodium alginate and yeast

This collection is the result of an investigation into the backwashing efficiency of polycarbonate (PC) membrane fouled by three types of organic foulants, protein, sodium alginate and yeast. In this experiement, polycarbonate (PC) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of polycarbonate (PC) membranes' fouling layer when three types of organic foulants (protein, sodium alginate and yeast) are present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers who are evaluating the backwashing efficiency of PC membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water..

Evaluating backwashing efficiency of polycarbonate (PC) membrane fouled by protein and yeast

This collection is the result of an investigation into the backwashing efficiency of polycarbonate (PC) membrane fouled by two types of organic foulants, protein and yeast. In this experiment, polycarbonate (PC) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of polycarbonate (PC) membranes' fouling layer when two types of organic foulants (protein and yeast) present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers evaluating the backwashing efficiency of PC membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water.

Evaluating backwashing efficiency of polycarbonate (PC) membrane fouled by sodium alginate and yeast

This collection is the result of an investigation into the backwashing efficiency of polycarbonate (PC) membrane fouled by two types of organic foulants, sodium alginate and yeast. In this experiement, polycarbonate (PC) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of polycarbonate (PC) membranes' fouling layer when two types of organic foulants (sodium alginate and yeast) present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers evaluating the backwashing efficiency of PC membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water..

Study on the fouling phenomena of poly(vinylidene fluoride) (PVDF) membrane by protein, yeast and sodium alginate

This sub-collection is the result of an investigation into the mechanism of organic fouling in membrane filtration processes. In this experiment, poly(vinylidene fluoride) (PVDF) membranes were used to filter three types of organic foulants, yeast, protein and sodium alginate with a concentration of 50mg/l, 40mg/l and 20 mg/l, respectively, from suspension in a dead-end filtration cell. These model foulants were stained with fluorescent dyes before filtration. This dataset contains a stack of images of the fouling layer on the PVDF membrane surface captured by a confocal laser scanning microscope (CLSM) and its associated acquisition software. This dataset would be useful to researchers who are investigating the membrane organic fouling mechanism so that new membrane materials and new anti-fouling surface treatment technologies can be developed for water and wastewater industry in the future .

Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm^-1 was represented by ν(C=O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C¹⁶ and C^18:1 with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids. 2012, The Society for Biotechnology, Japan. All rights reserved.