Re-programming of translation following cell stress allows IRES-mediated translation to predominate

There is now an overwhelming body of evidence to suggest that internal ribosome entry is required to maintain the expression of specific proteins during patho-physiological situations when cap-dependent translation is compromised, for example, following heat shock or during mitosis, hypoxia, differentiation and apoptosis. Translational profiling has been used by several groups to assess the extent to which alternative mechanisms of translation initiation selectively recruit mRNAs to polysomes during cell stress. The data from these studies have shown that under each condition 3-5% of coding mRNAs remain associated with the polysomes. Importantly, the genes identified in each of these studies do not show a significant amount of overlap, suggesting that 10-15% of all mRNAs have the capability for their initiation to occur via alternative mechanism(s).     

Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during data analysis.     

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation. Published in Russian in Biokhimiya, 2008, Vol. 73, No.9, pp. 1274–1280.     

Role of 5′‐ and 3′‐untranslated regions of mRNAs in human diseases

Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis‐regulatory elements, which are located in the 5′‐ and 3′‐UTRs (untranslated regions), and trans‐acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC‐richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis‐regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimer's disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.     

口蹄疫病毒基因组内部核糖体进入位点一级和二级结构分析

以口蹄疫病毒(foot-and-mouth disease virus,FMDV)强毒China/99株牛舌水泡皮为材料,用RT-PCR法提取RNA及扩增目的cDNA,然后与pGEM-T Easy载体连接并转化JM109菌株,再经重组质粒电泳、PCR和EcoRI酶切鉴定.用DNAstar软件比较了内部核糖体进入位点(IRES)的序列差异,并用RNAdraw软件绘制和分析了该区段的二级结构.8株FMDV IRES核苷酸序列比较表明该区段较为保守,并对非保守区域进行了分析.二级结构分析表明,FMDV IRES至少有3种二级结构图形:第一型有5个结构域,与Pilipenko等报道的一致;第二和三型分别有6和11个结构域,与Pilipenko等报道的结果不同.无论FMDV IRES二级结构如何不同,但单链区大部分核苷酸序列或基序相同,如AACUCC、GAAA、CUUU、AGG、AACC、GUAA等.茎环柄部核苷酸对维持二级结构的空间构像具有十分重要的作用,环中或单链区序列(基序)在维持其功能方面具有很重要的作用,如GAAA和CUUU基序分别是三级结构的组件和嘧啶区结合蛋白的结合位点.     

Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation

Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA_i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.     

An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non‐intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non‐stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA_i to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than‐average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.     

Life cycle assessment of electrical and thermal energy systems for commercial buildings

Background, Aim and Scope The objective of this life cycle assessment (LCA) study is to develop LCA models for energy systems in order to assess the potential environmental impacts that might result from meeting energy demands in buildings. The scope of the study includes LCA models of the average electricity generation mix in the USA, a natural gas combined cycle (NGCC) power plant, a solid oxide fuel cell (SOFC) cogeneration system; a microturbine (MT) cogeneration system; an internal combustion engine (ICE) cogeneration system; and a gas boiler. Methods LCA is used to model energy systems and obtain the life cycle environmental indicators that might result when these systems are used to generate a unit energy output. The intended use of the LCA analysis is to investigate the operational characteristics of these systems while considering their potential environmental impacts to improve building design using a mixed integer linear programming (MILP) optimization model. Results The environmental impact categories chosen to assess the performance of the energy systems are global warming potential (GWP), acidification potential (AP), tropospheric ozone precursor potential (TOPP), and primary energy consumption (PE). These factors are obtained for the average electricity generation mix, the NGCC, the gas boiler, as well as for the cogeneration systems at different part load operation. The contribution of the major emissions to the emission factors is discussed. Discussion The analysis of the life cycle impact categories indicates that the electrical to thermal energy production ratio has a direct influence on the value of the life cycle PE consumption factors. Energy systems with high electrical to thermal ratios (such as the SOFC cogeneration systems and the NGCC power plant) have low PE consumption factors, whereas those with low electrical to thermal ratios (such as the MT cogeneration system) have high PE consumption factors. In the case of GWP, the values of the life cycle GWP obtained from the energy systems do not only depend on the efficiencies of the systems but also on the origins of emissions contributing to GWP. When evaluating the life cycle AP and TOPP, the types of fuel as well as the combustion characteristics of the energy systems are the main factors that influence the values of AP and TOPP. Conclusions An LCA study is performed to eraluate the life cycle emission factors of energy systems that can be used to meet the energy demand of buildings. Cogeneration systems produce utilizable thermal energy when used to meet a certain electrical demand which can make them an attractive alternative to conventional systems. The life cycle GWP, AP, TOPP and PE consumption factors are obtained for utility systems as well as cogeneration systems at different part load operation levels for the production of one kWh of energy output. Recommendations and Perspectives Although the emission factors vary for the different energy systems, they are not the only factors that influence the selection of the optimal system for building operations. The total efficiencies of the system play a significant part in the selection of the desirable technology. Other factors, such as the demand characteristics of a particular building, influence the selection of energy systems. The emission factors obtained from this LCA study are used as coefficients of decision variables in the formulation of an MILP to optimize the selection of energy systems based on environmental criteria by taking into consideration the system efficiencies, emission characteristics, part load operation, and building energy demands. Therefore, the emission factors should not be regarded as the only criteria for choosing the technology that could result in lower environmental impacts, but rather one of several factors that determine the selection of the optimum energy system. ESS-Submission Editor: Arpad Horvath (horvath@ce.berkeley.edu)     

Statistical potentials for hairpin and internal loops improve the accuracy of the predicted RNA structure

RNA is directly associated with a growing number of functions within the cell. The accurate prediction of different RNA higher-order structures from their nucleic acid sequences will provide insight into their functions and molecular mechanics. We have been determining statistical potentials for a collection of structural elements that is larger than the number of structural elements determined with experimentally determined energy values. The experimentally derived free energies and the statistical potentials for canonical base-pair stacks are analogous, demonstrating that statistical potentials derived from comparative data can be used as an alternative energetic parameter. A new computational infrastructure—RNA Comparative Analysis Database (rCAD)—that utilizes a relational database was developed to manipulate and analyze very large sequence alignments and secondary-structure data sets. Using rCAD, we determined a richer set of energetic parameters for RNA fundamental structural elements including hairpin and internal loops. A new version of RNAfold was developed to utilize these statistical potentials. Overall, these new statistical potentials for hairpin and internal loops integrated into the new version of RNAfold demonstrated significant improvements in the prediction accuracy of RNA secondary structure.     

A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression.     

Faulty old ideas about translational regulation paved the way for current confusion about how microRNAs function

Despite a recent surge of reports about how microRNAs might regulate translation, the question has not been answered. The proposed mechanisms contradict one another, and none is supported by strong evidence. This review explains some deficiencies in the experiments with microRNAs. Some of the problems are traceable to bad habits carried over from older studies of translational regulation, here illustrated by discussing two models involving mRNA binding proteins. One widely-accepted model, called into doubt by recent findings, is the maskin hypothesis for translational repression of cyclin B1 in Xenopus oocytes. The second dubious model postulates repression of translation of ceruloplasmin by mRNA binding proteins. A big fault in the latter case is reconstructing the imagined mechanism before looking carefully at the real thing--a criticism that applies also to studies with microRNAs. Experiments with microRNAs often employ internal ribosome entry sequences (IRESs) as tools, necessitating brief discussion of that topic. A sensitive new assay reveals that many putative IRESs promote expression of downstream cistrons via splicing rather than internal initiation of translation. Recent claims about the biological importance of IRES-binding proteins--including suggestions that these proteins might serve as targets for cancer therapy--are not supported by any meaningful evidence. The bottom line is that older studies of mRNA binding proteins and putative IRESs have created a confusing picture of translational regulation which is not helpful when trying to understand how microRNAs might work. The obvious biological importance of microRNAs makes it essential to understand how they do what they do. Fresh ways of thinking and looking are needed.