Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe

Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct—we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD

Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter

Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.Published versio

A Sir2-Like Protein Participates in Mycobacterial NHEJ

In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria

Methodologies, technologies and strategies for acoustic streaming based Acoustofluidics

Acoustofluidics offers contact-free manipulation of particles and fluids, enabling their uses in various life sciences, such as for biological and medical applications. Recently, there have been extensive studies on acoustic streaming-based acoustofluidics, which are formed inside a liquid agitated by leaky surface acoustic waves (SAWs) through applying radio frequency signals to interdigital transducers (IDTs) on a piezoelectric substrate. This paper aims to describe acoustic streaming-based acoustofluidics and provide readers with an unbiased perspective to determine which IDT structural designs and techniques are most suitable for their research. This review, first, qualitatively and quantitatively introduces underlying physics of acoustic streaming. Then, it comprehensively discusses the fundamental designs of IDT technology for generating various types of acoustic streaming phenomena. Acoustic streaming-related methodologies and the corresponding biomedical applications are highlighted and discussed, according to either standing surface acoustic waves or traveling surface acoustic waves generated, and also sessile droplets or continuous fluids used. Traveling SAW-based acoustofluidics generate various physical phenomena including mixing, concentration, rotation, pumping, jetting, nebulization/atomization, and droplet generation, as well as mixing and concentration of liquid in a channel/chamber. Standing SAWs induce streaming for digital and continuous acoustofluidics, which can be used for mixing, sorting, and trapping in a channel/chamber. Key challenges, future developments, and directions for acoustic streaming-based acoustofluidics are finally discussed

Characterization of nonsense mediated mRNA decay in Schizosaccharomyces pombe

Nonsense mediated mRNA decay (NMD) is a translation-coupled process that preferentially destroys mRNAs harboring premature translation termination codons (PTCs). In mammalian cells, NMD is linked to pre-mRNA splicing. Typically, PTCs elicit strong NMD only if positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD. However, recent studies have questioned the importance of splicing and the EJC in NMD and instead they have proposed that, from yeast to mammalian cells, NMD is mostly determined by the distance of the PTC from the 3’ end. In this study, to investigate the link between pre-mRNA splicing and NMD, I used the fission yeast Schizosaccharomyces pombe. Many genes carry introns in fission yeast and unlike in Saccharomyces cerevisiae, the genome encodes for proteins homologous to EJC components. S. pombe is a powerful model organism which is easily genetically manipulated and we envisaged that studying NMD in this organism should facilitate the understanding of the molecular mechanism and in particular the link between NMD and splicing. During my PhD research, I have developed a versatile gene reporter system to study NMD; with it I discovered that splicing strongly enhances NMD in fission yeast and, surprisingly, that the EJC does not appear to be required. Unexpectedly, I found that splicing enhances NMD when the intron is either before or after the PTC, and furthermore, it does so only when the intron is close to the PTC. These observations suggest that the effect of splicing on NMD is direct and not a secondary consequence of splicing enhancing translation. Splicing is not an absolute requirement for NMD; I found that PTCs located early in the coding region could induce NMD even in the absence of a nearby intron. However, against the prediction of current models, I found no strong correlation with the distance of the PTC from the 3’ end. In summary, during my PhD a versatile system has been developed to study NMD in fission yeast and what I have observed challenges current NMD models and provides new mechanistic insights into NMD

Stress-constrained shell-lattice infill structural optimisation for additive manufacturing

This paper presents a numerical study on stress-constrained shell-lattice infill structural optimisation. This problem is inherently challenging for several reasons: (i) different stress measures have to be used for the solid shell and the porous lattice infill, and the two types of stress constraints make the problem extremely complex to solve and (ii) involvement of the shell layer further complicates the optimisation problem modelling and its solution. To address these challenges, two stress constraints were formulated, i.e. a von Mises stress-based constraint for the solid shell layer and a Tsai-Hill yield criteria-based constraint for the porous lattice. Then, level set function is adopted to represent the shape of the shell layer and the constant-thickness shell layer is modelled based on the signed distance feature. Then, a comprehensive and accurate sensitivity result is derived to guide the concurrent structural shape and lattice density optimisation. A few numerical examples will be studied to prove the effectiveness of the developed algorithm. Some interesting phenomena have been observed, such as the soft narrow band at the shell-lattice interface to mitigate stress concentration