Legal Implications of the Use of Export Taxes in Addressing Carbon Leakage: Competing Border Adjustment Measures

The prevailing uncertainties about the future of the post-Kyoto international legal framework for climate mitigation and adaptation increase the likelihood of unilateral trade interventions that aim to address climate policy concerns, as exemplified by the controversial European Union initiative to include the aviation industry in its emissions trading system. The emerging literature suggests that border carbon adjustment (BCA) measures imposed by importing countries would lead to substantial legal complications in relation to World Trade Organization law and hence to possible trade disputes. Lack of legal clarity on BCAs is exacerbated by potential counter or pre-emptive export restrictions that exporting countries might impose on carbon-intensive products. In this context, this paper investigates the interface between legal and welfare implications of competing unilateral BCA measures. It argues that carbon export taxes will be an inevitable part of the future climate change regime in the absence of a multilateral agreement. It also describes the channels through which competing BCAs may lead to trade conflicts and political complications as a result of their distributional and welfare impacts at the domestic and global levels.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

An mRNA-Derived Noncoding RNA Targets and Regulates the Ribosome.

The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.

Modeling Responses of Diatom Productivity and Biogenic Silica Export to Iron Enrichment in the Equatorial Pacific Ocean

Using a three-dimensional physical-biogeochemical model, we have investigated the modeled responses of diatom productivity and biogenic silica export to iron enrichment in the equatorial Pacific, and compared the model simulation with in situ (IronEx II) iron fertilization results. In the eastern equatorial Pacific, an area of 540,000 km(2) was enhanced with iron by changing the photosynthetic efficiency and silicate and nitrogen uptake kinetics of phytoplankton in the model for a period of 20 days. The vertically integrated Chl a and primary production increased by about threefold 5 days after the start of the experiment, similar to that observed in the IronEx II experiment. Diatoms contribute to the initial increase of the total phytoplankton biomass, but decrease sharply after 10 days because of mesozooplankton grazing. The modeled surface nutrients (silicate and nitrate) and TCO(2) anomaly fields, obtained from the difference between the "iron addition'' and "ambient'' (without iron) concentrations, also agreed well with the IronEx II observations. The enriched patch is tracked with an inert tracer similar to the SF6 used in the IronEx II. The modeled depth-time distribution of sinking biogenic silica (BSi) indicates that it would take more than 30 days after iron injection to detect any significant BSi export out of the euphotic zone. Sensitivity studies were performed to establish the importance of fertilized patch size, duration of fertilization, and the role of mesozooplankton grazing. A larger size of the iron patch tends to produce a broader extent and longer-lasting phytoplankton blooms. Longer duration prolongs phytoplankton growth, but higher zooplankton grazing pressure prevents significant phytoplankton biomass accumulation. With the same treatment of iron fertilization in the model, lowering mesozooplankton grazing rate generates much stronger diatom bloom, but it is terminated by Si(OH)(4) limitation after the initial rapid increase. Increasing mesozooplankton grazing rate, the diatom increase due to iron addition stays at minimum level, but small phytoplankton tend to increase. The numerical model experiments demonstrate the value of ecosystem modeling for evaluating the detailed interaction between biogeochemical cycle and iron fertilization in the equatorial Pacific.

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: A comparison of tricyclo-DNA with LNA

The nuclear antisense properties of a series of tricyclo(tc)-DNA oligonucleotide 9-15mers, targeted against the 3' and 5' splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence and dose dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by a factor of at least 4-5. A tc-oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2‘M concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction of CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc-15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc- and LNA-oligonucleotides. The obtained results confirm the power of tricyclo-DNA for nuclear antisense applications. Morover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.