PAXX and its paralogues synergistically direct DNA polymerase λ activity in DNA repair

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5′ end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.This work was supported by the Medical Research Council (MRC) UK

eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR

BackgroundRegulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood.ResultsHere, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5′UTR of target mRNAs directly upstream of the AUG start codon.ConclusionsOur data support a model whereby purine motifs towards the 3′ end of the 5′UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding.</p

eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5'UTR

Background: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Results: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5′UTR of target mRNAs directly upstream of the AUG start codon. Conclusions: Our data support a model whereby purine motifs towards the 3′ end of the 5′UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding

Characterisation of the -700kDa apoptosome complex from THP.1 cells

Apoptosis or programmed cell death is a process involving the regulated self-destruction of a cell. Execution of the apoptotic programme requires the hierarchical activation of a family of proteases, the caspases. Here, I describe the characterisation and purification of the apoptosome, a ~700kDa caspase activating complex. Gel filtration of dATP-activated THP.1 cell lysates showed that effector caspase activity is assessed with a large complex, the apoptosome, which contains Apaf-1 and active caspases-9, -3 and -7. Further analysis revealed two large complexes, a biologically inactive ~1.4MDa and a biologically active ~700kDa complex to be formed very rapidly. Experiments involving the use of the caspase inhibitor z-VAD.FMK demonstrated that complex formation is caspase-independent, however, release of processed caspase-9 from the complex and the recruitment of other procaspases to the complex, is caspase activity-dependent. In apoptotic cells, the ~700kDa apoptosome complex predominates and is therefore suggested to be the biologically relevant complex. Normal intracellular K+ concentrations inhibited formation of the ~700kDa apoptosome complex in lysates and in a reconstituted system. Increasing concentrations of cytochrome c partially reversed inhibition. A mechanism is proposed for the inhibitory effect of K+ during dATP-dependent caspase activation in cellular lysates. The ~700kDa apoptosome complex was purified using multi-step purification procedures. Several proteins were identified in the purified apoptosome fraction and these included Apaf-1 and capase-9 as well as XIAP, b-actin, CAP-1, CAZ1/CAZ2 and possibly Hsc70 are all involved with the cytoskeleton and are capable of binding actin, whereas rabaptin-5 and IKKg (NEMO) are known to be involved in endosomal transport and the NFkB pathway, respectively. The possible involvement of these proteins in apoptosome assembly and function is discussed

Development of a versatile rheocasting technology

Since 2003, a concerted effort between Alcan Inc. (ARDC: Arvida Research and Development Centre) and the Aluminium Technology Centre (ATC) of the National Research Council of Canada is underway to develop a technology, dubbed SEED (Swirled Enthalpy Equilibration Device), to produce semi-solid aluminium feedstock. This technology, patented by Alcan Inc., is a simple process offering many advantages over thixocasting, especially for reducing the cost of feedstock. The process involves two main steps: 1) heat extraction to achieve a desired liquid/solid mixture, and 2) drainage of an excess liquid to produce a self-supporting semi-solid slug that is cast in a high pressure press. This paper reports that the SEED technology is applicable to a number of aluminium alloys and can be easily adapted to produce a wide range of slug dimensions. Furthermore, since the heat transfer plays a predominant role during the manufacture of the semi-solid slurry, its analysis is also presented.Peer reviewed: YesNRC publication: Ye

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DEAD-box helicase eIF4A2 inhibits CNOT7 deadenylation activity

The CCR4–NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4–NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4–NOT complex but instead inhibits CNOT7 activity. We identified a CCR4–NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4–NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4–NOT complex which results in different pathways for translational repression and mRNA deadenylation

A systematic approach for testing expression of human full-length proteins in cell-free expression systems

Abstract Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.</p