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Cohomologie ????-adique de la tour de Drinfeld: le cas de la dimension 1

Pierre Colmez, Gabriel Dospinescu and Wiesława Nizioł
J. Amer. Math. Soc. 33 (2019), 311-362.
Abstract, references and article information




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A PP2A Phosphatase High Density Interaction Network Identifies a Novel Striatin-interacting Phosphatase and Kinase Complex Linked to the Cerebral Cavernous Malformation 3 (CCM3) Protein

Marilyn Goudreault
Jan 1, 2009; 8:157-171
Research




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Analysis of the Subunit Composition of Complex I from Bovine Heart Mitochondria

Joe Carroll
Feb 1, 2003; 2:117-126
Research




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Lysine Acetylation Is a Highly Abundant and Evolutionarily Conserved Modification in Escherichia Coli

Junmei Zhang
Feb 1, 2009; 8:215-225
Research




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Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Amelia C. Peterson
Nov 1, 2012; 11:1475-1488
Technological Innovation and Resources




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Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation

Boris Macek
Feb 1, 2008; 7:299-307
Research




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In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy

Ivan Matic
Jan 1, 2008; 7:132-144
Research




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Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

Jonathan C. Trinidad
Aug 1, 2012; 11:215-229
Research




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Complementary Profiling of Gene Expression at the Transcriptome and Proteome Levels in Saccharomyces cerevisiae

Timothy J. Griffin
Apr 1, 2002; 1:323-333
Research




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A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions: Application in Ubiquitin Profiling and Protein Complex Identification Combined with in vivoCross-Linking

Christian Tagwerker
Apr 1, 2006; 5:737-748
Research




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Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics

William M. Old
Oct 1, 2005; 4:1487-1502
Research




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Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins

Leigh Anderson
Apr 1, 2006; 5:573-588
Research




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The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra

Ignat V. Shilov
Sep 1, 2007; 6:1638-1655
Technology




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Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns

Martin R. Larsen
Jul 1, 2005; 4:873-886
Technology




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Analysis of the Human Tissue-specific Expression by Genome-wide Integration of Transcriptomics and Antibody-based Proteomics

Linn Fagerberg
Feb 1, 2014; 13:397-406
Research




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Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

Philip L. Ross
Dec 1, 2004; 3:1154-1169
Research




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The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus [Protein Synthesis and Degradation]

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.




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SUMOylation of the transcription factor ZFHX3 at Lys-2806 requires SAE1, UBC9, and PIAS2 and enhances its stability and function in cell proliferation [Protein Synthesis and Degradation]

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.




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Heterotrimeric Gq proteins as therapeutic targets? [Molecular Bases of Disease]

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.




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Noncatalytic Bruton's tyrosine kinase activates PLC{gamma}2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells [Membrane Biology]

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.




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G{alpha}q splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila [Signal Transduction]

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein–coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, Gαq1 null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the Gαq gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in Gαq1 null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis.




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NF-{kappa}B mediates lipopolysaccharide-induced alternative pre-mRNA splicing of MyD88 in mouse macrophages [Signal Transduction]

Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled.




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Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila [Signal Transduction]

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.




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DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels [Cell Biology]

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.




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Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation [Immunology]

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.




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Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2 [Genomics and Proteomics]

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein–coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.




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Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells [Signal Transduction]

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11–7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.




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Five Foreign Policy Questions for the UK’s Next Prime Minister

18 June 2019

Thomas Raines

Director, Europe Programme
Even if most don’t get to vote in the Conservative leadership election, the public deserves serious answers on the foreign policy plans of those who want to lead the country.

2019-06-18-10D.jpg

10 Downing Street. Photo: Getty Images.

In a month’s time, the UK will have a new prime minister. The campaign has been dominated by candidates’ views on how to deliver Britain’s withdrawal from the EU by October, alongside some discussion of domestic issues.

But relatively little has been said about international affairs, despite the fact that foreign policy questions are becoming a more partisan issue and Britain is facing crucial questions around Brexit and the wider international context. These will be waiting for whoever finds themselves in Number 10 on 22 July. 

1. How can Britain influence Europe from outside the EU?

Theresa May was fond of saying that Britain is leaving the EU but not leaving Europe. Britain cannot change its geography. It will continue to share many strategic and security interests with the rest of the continent, but it will be outside of Europe’s central political and economic project. A new PM will not only have to negotiate Britain’s new relationship with the EU, but also think about how to influence it as a third country.

An aspect of this will be finding a new way to work with the EU on foreign, security and defence policy that meets the need for autonomy on both sides. How deep this relationship is or how institutionalized it will be is yet to be negotiated and can be shaped by the next PM.

The UK needs to decide how ambitiously it wants to engage with the new defence agenda in the EU (particularly its industrial components), and how to balance these with key bilateral relationships like France and Germany. Amid uncertainty about American security guarantees and Russian aggression, the next PM must also consider what Britain’s security role in Europe should be and NATO’s place within that.

Beyond conventional foreign policy issues, Britain is also going to be heavily shaped and influenced by the rule-making power of the EU, and how the world’s largest market regulates itself, from energy to financial services, consumer products and the environment.

The UK will need a strategy to influence the EU from the outside – something Swiss, Norwegians and Americans will acknowledge is no easy feat. This could include significantly increasing its diplomatic footprint across Europe, working closely with the UK’s private and non-profit sectors, utilizing Britain’s technical expertise in areas like sanctions, and creating new ad hoc groupings to share perspectives and ideas, building on examples like the Northern Future Forum

2. Should Britain do business with President Trump?

President Trump represents a fundamental challenge to Britain: an American president whose belligerent unilateralism runs counter to many of Britain’s foreign policy objectives. His frequent and often shameless diplomatic faux pas – from proposing Nigel Farage be the British ambassador to his dog-whistle attacks on the mayor of London – are compounded by real differences of substance on issues like trade, climate change and nuclear non-proliferation.

A new prime minister must decide how to manage relations with the US administration, whether to challenge or condemn a US president when he acts against Britain’s interests, or use flattery or quieter diplomacy to seek to influence him. Theresa May’s strategy of staying politically close to the president and playing to his ego has yielded little in policy terms, though other world leaders have fared little better.

A new PM will face some uncomfortable choices. Will they continue to defend the Iran nuclear deal alongside European allies while the US continues to undermine it? Do they believe a trade deal with America is desirable or achievable with the current administration, and what are they willing to sacrifice to achieve it? Is the American security guarantee for Europe secure with Donald Trump as president? Judgements on these questions should inform Britain's wider strategy, and its objectives for a future relationship with the European Union.

3. Should Britain prioritize economics or security in its relations with China?

Britain faces its own version of the challenge that many countries face – how to balance the economic and investment benefits of a positive relationship with China with concerns about repressive domestic politics and a more assertive Chinese role regionally and globally. This tension has become more acute for two reasons.

First, the economic dislocation of leaving the EU may create a greater reliance on Chinese trade and investment. China is already a major investor in the UK. If Brexit proves to be disorderly, Britain’s need may be all the greater (though China faces economic headwinds as well). Some in Brussels even fear that the economic difficulties of Brexit may make the UK a soft touch for emerging powers from which it seeks inward investment and market access.

Second, the deterioration in US–China relations means the UK may come under increasing pressure from the United States to take a tough line with China. The controversy over Huawei’s role in delivering 5G networks may become a more regular feature of transatlantic debates, with Britain facing Chinese economic pressure on one side and a squeeze from America over security issues on the other, without the weight of the EU behind it.  

A new prime minister should consider whether the UK’s interests are served by a security role in east Asia, and whether it has the capability to play one.

The UK remains a party to the Five Power Defence Arrangements. The Royal Navy has conducted freedom of navigation exercises in the South China Sea, prompting a rebuke from Beijing. It has also taken steps to deepen security ties with Japan.

But the UK government has struggled to present a coherent position. Some cabinet ministers have sought to open doors to the Chinese market at the same time as others announced their intentions to send aircraft carriers to the Pacific. The next PM will need to find a balance between China and the US, or accept the consequences of more directly taking sides on disputes about trade, technology, and security.

4. How can the contradictions between UK foreign and domestic policy be reconciled?

One of the many problems with the vague and unhelpful slogan ‘Global Britain’ is how it jars with many aspects of domestic policy. This incoherence reduces Britain’s foreign policy credibility and effectiveness.

Britain has actively supported the UN-led Yemen peace process while continuing to support Saudi Arabia’s military campaign through arms sales. Britain wants to build a new ambitious independent trade policy while restricting the migration that is crucial for services trade. British foreign secretaries trumpet the UK’s soft power while the Home Office deports members of the Windrush generation, bungles EU citizenship applications and sets unreasonable burdens for many people seeking visas simply to visit the country.

Global universities are celebrated while international students had their post-study visas cut (a policy that sensibly is likely to be reversed). Britain advocates international tax compliance and transparency while not taking robust steps to regulate the tax haven role played by crown dependencies and overseas territories.

A new prime minister has the chance to get to grips with these inconsistencies and develop foreign and domestic policies which are more coherent and self-re-enforcing.

5. At what level should Britain’s international ambitions be funded?

Successive governments have celebrated the fact the UK is the only Western country to spend 2% of GDP on defence and 0.7% on development. However, this masks some real pressures in the system.

There are significant problems in the defence budget and a growing gap between commitments and committed funds. Meanwhile, the funding of Britain’s diplomacy has been cut by successive governments – Labour, Conservative and coalition – for much of the last 20 years. Numerous bodies have highlighted the problems facing the overstretched and underfunded Foreign Office. Where would defence and diplomacy sit in the new prime minister’s hierarchy of priorities?

The problem is not purely one of funding, but the gap between ambitions, rhetoric and resources. It is not sustainable for British ministers to trumpet Britain’s global ambitions while not properly funding the tools of its influence abroad.

It would be reasonable and understandable for a new prime minister to adjust that ambition and tone down the rhetoric, or alternatively to address resource pressures by investing in diplomacy and defence. But that choice should be informed by a sober reflection on Britain’s international position and interests as it leaves the EU. Brexit offers a chance to revisit assumptions that have guided British policy for a generation. A new prime minister should seize this opportunity.

A realistic vision for the future

All these issues will be more pronounced if the UK leaves the EU with no deal at the end of October. ‘No deal’ would be not simply an economic shock but a diplomatic rupture that will colour the UK’s capacity to negotiate a new relationship with the EU, which will be the first order of business after a ‘no deal’ exit. Trust will be in short supply.

Even if they don’t get to vote on the new prime minister, the public deserves serious answers to these and other questions from the men who want to lead the country. Not the platitudes of ‘Global Britain’ or a reflexive and unexamined British exceptionalism, but a serious, realistic assessment of how Britain will cope with the disruptions of leaving the EU and how it might thrive outside the regional bloc it has been a part of for more than 45 years.




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EU Security Ambitions Are Hostage to the Brexit Process

25 June 2019

Professor Richard G Whitman

Associate Fellow, Europe Programme
The EU faces a fundamental contradiction in its goals to become more strategically autonomous in defence matters.

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Soldiers of a Eurocorps detachment raise the EU flag at the European Parliament in Strasbourg. Photo: Getty Images.

Three years ago, as the UK was holding its referendum on Brexit, the EU was rolling out its Global Strategy for a more cohesive and effective security and defence policy. Since then, EU member states have set impressive goals and, as significantly, taken important practical steps to make an EU defence capability a tangible proposition, despite differing collective defence commitments, traditions of neutrality among some member states and very different strategic cultures.

All of these developments have taken place with the UK as reluctant observer. The UK has been traditionally hostile to a deepening of defence collaboration within the EU (and consistent of the view that Europe’s military security was best provided through NATO). But the Brexit referendum vote has placed the UK as a bystander as EU security and defence initiatives have been pursued which have overridden the past red lines of British governments.

There is, however, a Brexit-related paradox in all these developments.

A central goal of the security and defence-related aspects of the EU Global Strategy is for the EU to have the capacity to act independently of the United States and, through indigenous defence industries, the ability to produce the means to make that possible.

With the UK outside the EU, and its opposition absent, it is easier to create a political consensus to push for more defence integration. But without the UK there are diminished collective defence capabilities which would make European strategic autonomy much harder to achieve.  

The May government has been an enthusiast for preserving close security and defence cooperation with the EU. The Withdrawal Agreement and the Political Declaration both seek to provide for a close EU–UK relationship post-Brexit.

However, the Article 50 negotiations have made clear that the EU’s institutions are hostile to special treatment for the UK beyond that normally accorded to a third country. Disagreements over the terms of the UK’s continuing participation in the Gailleo dual-use satellite system, which has a significant security and defence utility, have signalled that there is a strong lobby in Brussels and some national capitals seeking to significantly circumscribe collaboration with Britain.

The scale and capabilities of the UK’s military, its defence expenditure (notably on defence research and development) and its defence industrial base make any British decoupling from the EU’s security and defence a major issue. Disconnecting the UK from EU developments entirely would be a costly political choice for both sides.

And excluding the UK from new initiatives in defence R&D and new defence procurement arrangements would be suboptimal in delivering a stronger European defence, technological and industrial base. Duplicating existing UK capabilities, especially strategic enablers such as strategic airlift, target acquisition and intelligence, surveillance and reconnaissance capabilities, would be an unnecessary squandering of already hard-pressed European defence budgets.

At present the common procurement and defence industry plans driven by the EU Global Strategy are embryonic. And significant defence capability decisions are taking place detached from the EU’s plans, which could reinforce a divide between the UK and other member states.

As illustrative, the formal agreement this week between France, Germany and Spain on the Future Combat Air System (FCAS) to develop a next-generation stealth fighter is competing with the UK-supported Tempest project that shares the same objective. The 20-year timescales for the delivery of the FCAS and Tempest projects are a reminder that defence procurement decisions are of multi-decade significance.

As the EU’s ambitions are nascent, it is too early to fully assess what might be the impact of any decision by the EU and the UK to keep each other at an arms-length in security and defence cooperation. With a more detached relationship, the UK will have significant concerns if it sees the EU’s common procurement arrangements develop in a manner that actively discriminates against the UK defence industry.

Outside of procurement and defence issues there may be other areas of future concern for the UK – for example, the extent to which the EU might deepen and broaden cooperation with NATO in a manner that makes the collective influence of EU member states within NATO more apparent, or to which the footprint of future EU conflict and security activities in third countries starts to overshadow the activities of the UK.

As the UK has been grappling with Brexit domestically, the EU has been evolving its security and defence policy ambitions. These are developments that will impact on the UK and in which, therefore, it has a stake but as a departing member state it has a weakening ability to shape.

Any aspect of future EU–UK cooperation is hostage to the vagaries of how the Brexit endgame concludes.




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Brexit, Party Politics and the Next Prime Minister

Invitation Only Research Event

15 July 2019 - 8:30am to 9:30am

Chatham House | 10 St James's Square | London | SW1Y 4LE

Event participants

Daniel Finkelstein OBE, Associate Editor, The Times; Conservative Member of the House of Lords; Chairman, Onward 
Chair: Thomas Raines, Head, Europe Programme, Chatham House

With the new leader of the Conservative party due to be announced on 23 July, what are the prospects for the party and the country?

On Brexit, the new prime minister faces most of the same challenges and constraints as Theresa May. The leadership contenders have outlined their ambitions for a revised deal, but with the EU insisting negotiations are over, their room for manoeuvre appears to be limited. Furthermore, even with a new leader at the helm, important divisions remain among voters about what shape Brexit and the future UK-EU relationship should take. If the EU won’t change the deal, and parliament won’t accept it, how can the deadlock be broken? Is a 'No Deal' Brexit politically deliverable? Or could there be a general election later in 2019? Can the Conservative party survive a pre-Brexit election intact?

Beyond Brexit, what are the other choices, in both domestic and foreign policy, facing the next prime minister? How might the decisions he makes affect the future of the party and British politics more broadly?

In this session, the speaker will share his reflections on the likely result of the leadership election, and what lies beyond it.

Attendance at this event is by invitation only.

Event attributes

Chatham House Rule

Alina Lyadova

Europe Programme Coordinator




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The Rise of China and the Future of the Transatlantic Relationship

29 July 2019

The growth of China's wealth and military power represents an epochal change in international politics. This briefing argues that China’s rise has worrying implications for the liberal international order and explores how this will affect the transatlantic relationship.

Jennifer Lind

Associate Fellow, US and the Americas Programme and Asia-Pacific Programme (based in the US)

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Teams from France, Great Britain, the US, China, Australia and Japan race against each other during the SailGP on 4 May 2019 in San Francisco, California. Photo: Getty Images

Summary

  • The stakeholders in the transatlantic relationship – the US, Canada and Europe – have long sought to stabilize international politics and economies by spreading support for the liberal goals of free markets, democracy and human rights. As their own commitment to this agenda appears to waver, China is becoming wealthier and more assertive. This briefing explores the extent to which these goals – along with the unity of the transatlantic relationship – are now in jeopardy.
  • Great uncertainty surrounds this question, including over the direction of US foreign policy, risks to European cohesion and slowing growth in China. However, two decades of revisionist behaviour by the authorities in Beijing show that China’s values and interests already conflict with transatlantic goals in trade, cyberspace, international development, security and human rights.
  • On trade, China pursues protectionist policies while engaging actively in intellectual property theft. China’s military modernization and its view of maritime law challenge the territorial status quo in East Asia and raise the risk of military crisis there. China lends unconditionally to countries that abuse human rights and are corrupt, undermining efforts by Western governments to promote good governance and human rights.
  • Defending liberal goals is complicated by asymmetric interests among the transatlantic partners, especially over security. China also uses ‘wedge’ strategies to pick off potential allies, thus diluting the power and will of any counterbalancing effort.
  • This briefing argues that China’s rise has worrying implications for the liberal international order. In response, the US should recognize its own strong interest in European unity, while Europeans must be ready to align more with the US (and East Asian allies) in order to temper Chinese behaviour.




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The Future of Brexit, Britain and Boris

Invitation Only Research Event

17 October 2019 - 8:30am to 9:30am

Chatham House | 10 St James's Square | London | SW1Y 4LE

Event participants

Philip Rycroft, Permanent Secretary, Department for Exiting the EU (2017-19)
Chair: Tom Raines, Head, Europe Programme, Chatham House

Once again, Brexit is reaching an important juncture. The European Council on 17 October is seen by many as the last chance for Britain to secure a withdrawal deal or another delay. With parliament and government at loggerheads, it is far from clear what happens at the end of October.
 
In a discussion taking place on the day of the European Summit, the speaker will share his insights on the future of negotiations between the UK and the EU under different scenarios. How do the EU and the UK repair relations after 'No Deal'? Should a withdrawal deal be agreed, what does the second phase of negotiations look like and what lessons can Whitehall draw from phase one? And if there is another delay, what will change over the course of the next three months?
 
Please note attendance at this event is by invitation only and places are now full. 

Event attributes

Chatham House Rule

Alina Lyadova

Europe Programme Coordinator




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Britain, the EU and the Power of Myths: What Does Brexit Reveal about Europe?

Invitation Only Research Event

14 November 2019 - 8:30am to 9:30am

Chatham House | 10 St James's Square | London | SW1Y 4LE

Event participants

Kalypso Nicolaïdis, Professor of International Relations, Faculty Fellow, St Antony’s College, University of Oxford; Author, Exodus, Reckoning, Sacrifice: Three Meanings of Brexit
Chair: Hans Kundnani, Senior Research Fellow, Europe Programme, Chatham House

When we look back on Brexit, what will it tell us about Europe? Will it simply be that an insular UK was always different and destined to never fit in? Will it be that the UK's decision to leave revealed deeper problems with the EU? Or will it be that the threat created by the UK's withdrawal united the continent and saved the European project?

The speaker will explore Brexit through the prism of biblical and ancient Greek mythology. She will examine the reasons behind Britain’s decision to leave the EU and imagine a ‘better Europe’ that has learnt the lessons of the past and reconciled the divisions exposed by the Brexit vote. How can the EU reinvent itself and re-engage its citizens? And where does a post-Brexit UK fit?

Attendance at this event is by invitation only. 

Event attributes

Chatham House Rule

Alina Lyadova

Europe Programme Coordinator




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Why Britain’s 2019 Election Is Its Most Unpredictable in Recent History

7 November 2019

Professor Matthew Goodwin

Visiting Senior Fellow, Europe Programme
Leadership concerns and a collapse of traditional party loyalties make the December vote uncommonly volatile.

On 12 December, Britain will hold the most consequential election in its postwar history. The outcome of the election will influence not only the fate of Brexit but also the likelihood of a second referendum on EU membership, a second independence referendum in Scotland, the most economically radical Labour Party for a generation, Britain’s foreign and security policy and, ultimately, its position in the wider international order.

If you look only at the latest polls, then the outcome looks fairly certain. Ever since a majority of MPs voted to hold the election, the incumbent Conservative Party has averaged 38%, the opposition Labour Party 27%, the Liberal Democrats 16%, Brexit Party 10%, Greens 4% and Scottish National Party 3%. Prime Minister Boris Johnson and his party continue to average an 11-point lead which, if this holds until the election, would likely deliver a comfortable majority.

Johnson can also point to other favourable metrics. When voters are asked who would make the ‘best prime minister’, a clear plurality (43%) say Johnson while only a small minority (20%) choose the Labour Party leader, Jeremy Corbyn. Polls also suggest that, on the whole, Johnson is more trusted by voters than Corbyn to deal with Brexit, the economy and crime, while Jeremy Corbyn only tends to enjoy leads on health. All of this lends credence to the claim that Britain could be set for a Conservative majority and, in turn, the passing of a withdrawal agreement bill in early 2020.

But these polls also hide a lot of other shifts that are taking place and which, combined, make the 2019 general election unpredictable. One concerns leadership. While Boris Johnson enjoys stronger leadership ratings than Jeremy Corbyn, it should be remembered that what unites Britain’s current generation of party leaders is that they are all unpopular. Data compiled by Ipsos-MORI reveals that while Johnson has the lowest ratings of any new prime minister, Labour’s Jeremy Corbyn has the lowest ratings of any opposition leader since records began.

Another deeper shift is fragmentation. One irony of Britain’s Brexit moment is that ever since the country voted to leave the European Union its politics have looked more ‘European’. Over the past year, one of the world’s most stable two-party systems has imploded into a four-party race, with the anti-Brexit Liberal Democrats and Nigel Farage’s strongly Eurosceptic Brexit Party both presenting a serious challenge to the two mainstream parties.

In the latest polls, for example, Labour and the Conservatives are attracting only 61 per cent of the overall vote, well down on the 80 per cent they polled in 2017. Labour is weakened by the fact that it is only currently attracting 53 per cent of people who voted Labour at the last election, in 2017. A large number of these 2017 Labour voters, nearly one in four, have left for the Liberal Democrats, who are promising to revoke Article 50 and ‘cancel Brexit’. This divide in the Remain vote will produce unpredictable outcomes at the constituency level.

At the other end of the spectrum, the Conservatives are grappling with a similar but less severe threat. Nigel Farage and the Brexit Party are attracting around one in ten people who voted Conservative in 2017, which will make Boris Johnson’s task of capturing the crucial ‘Labour Leave’ seats harder. There is clear evidence that Johnson has been curbing Farage’s appeal, but it remains unclear how this rivalry on the right will play out from one seat to the next.

One clue as to what happens next can be found in those leadership ratings. While 80 per cent of Brexit Party voters back Johnson over Corbyn, only 25 per cent of Liberal Democrat voters back Corbyn over Johnson. Johnson may find it easier to consolidate the Leave vote than Corbyn will find the task of consolidating the Remain vote.

All of this reflects another reason why the election is unpredictable: volatility. This election is already Britain’s fifth nationwide election in only four years. After the 2015 general election, 2016 EU referendum, 2017 general election and 2019 European parliament elections, Britain’s political system and electorate have been in a state of almost continual flux. Along the way, a large number of voters have reassessed their loyalties.

As the British Election Study makes clear, the current rate of ‘vote-switching’ in British politics, where people switch their vote from one election to the next, is largely unprecedented in the post-war era. Across the three elections held in 2010, 2015 and 2017, a striking 49 per cent of people switched their vote.

This is not all about Brexit. Attachment to the main parties has been weakening since the 1960s. But Brexit is now accelerating this process as tribal identities as ‘Remainers’ or ‘Leavers’ cut across traditional party loyalties. All this volatility not only gives good reason to expect further shifts in support during the campaign but to also meet any confident predictions about the election result with a healthy dose of scepticism.




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UK General Election 2019: Britain's New Foreign Policy Divide

9 December 2019

Thomas Raines

Director, Europe Programme
A breakdown of foreign policy consensus means voters have a meaningful choice between two different visions of Britain’s place in the world.

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Boris Johnson and Jeremy Corbyn at the state opening of Parliament in October. Photo: Getty Images.

Genuine ideological differences have returned to British politics. That is as true in foreign policy as in questions of domestic politics. The post-Cold War foreign policy consensus in UK politics around liberal multilateralism is fraying.

This tradition had some key characteristics. It saw Britain as one of the cornerstones of an international order built on a liberal (or neo-liberal if you prefer) approach to economic globalization. EU membership was considered central to Britain’s influence and prosperity (even if further political integration never had deep support). Security policy was grounded in a stable package of NATO membership, close ties to the US, nuclear deterrence and a willingness to conduct military intervention.

Both main parties accepted that foreign policy had a commercial dimension. Both were willing to sell arms abroad to regimes with dubious domestic records.

Despite differences of emphasis, and some moments of genuine disagreement, foreign policy did not undergo big shifts as different parties traded periods in office. That may be set to change. 

Party divides

On the one hand, Labour wants to reset and re-orientate Britain’s international role based on human rights and international law. It promises a new internationalism and to end what it glibly calls the ‘bomb first, talk later’ approach, alongside a human rights-driven trade policy. More concretely, it promises to legislate to ensure Parliament takes decisions on military action, boost resources for the underfunded Foreign Office and suspend arms sales to Saudi Arabia for use in Yemen.  

In Jeremy Corbyn, they have a leader with roots in a distinct left-wing ideological tradition of internationalism that blends a commitment to international solidarity alongside anti-imperial and anti-war sentiment. He has spent his career as a sharp critic of Israeli and US policy, while championing various international political causes, some more radical or fringe than others. His historic positions on issues like NATO and nuclear deterrence, while not represented in the party manifesto, demonstrate a personal radicalism that no recent Labour PM has embodied.

His willingness to challenge the failures of the hitherto centre ground of foreign policy – particularly on military interventions from Iraq to Libya – is an under-appreciated aspect of his appeal among many supporters, even while it is one of the sharpest lines of attack from his critics. Boris Johnson’s chauvinistic rhetoric could not stand in sharper contrast to Labour’s commitment to conduct an audit of the effect of Britain’s colonial legacy on violence and insecurity.  

The Conservative manifesto asserts their pride in Britain’s historical role in the world, followed by a broad set of largely rhetorical commitments to bolster alliances and expand influence. An ambitious free trade agenda points to a more economic and commercially driven foreign policy, the inevitable trade-offs and constraints of which are only beginning to be addressed and debated.

There is an underlying sense that Britain will be liberated from the constraints of EU membership, although beyond trade there is little that would not have been possible, or in most cases easier, from within the EU. As my colleague Richard Whitman has observed, the empty bromide ‘Global Britain’ has been dropped altogether, though beyond the idea of a new UK space command and a stronger sanctions regime, there is little that is new or specific.  

Not all the consensus centre-ground position has been abandoned. Both major parties remain committed to spending 2 per cent of GDP on defence and 0.7 per cent of gross national income on development in their 2019 manifestos.

But beyond their manifesto commitments, prime ministers can exercise extensive powers in foreign affairs through the royal prerogative. Their government can choose to recognize other states, as Labour intends to do with Palestine. They can sign international treaties. And at present, in the absence of the sort of war powers act proposed by Labour, they can conduct military action without recourse to Parliament, which has no legally established role in this area.

Even a weak minority government would have considerable scope to transform the tone of Britain’s diplomacy.

Foreign policy as a partisan political issue

If UK foreign policy becomes more partisan, this will have longer term implications. Voters will theoretically have greater scope to shape and influence foreign policy more directly. Foreign policy may become divisive if it becomes more partisan. It may also become less consistent, which will affect the capacity of the UK to show leadership over the longer term on issues on which there is no domestic consensus. Britain’s allies may need to manage a less reliable partner. The diplomatic and security apparatus of Whitehall will need to be more adaptable.  

British elections generally don’t turn on foreign policy questions; 2019 will not buck that trend. At the same time, this election will be very influential in shaping Britain’s position on the world stage and its approach to international issues. Boris Johnson and Jeremy Corbyn represent very different ideas about Britain’s role: its foreign policy, its alliances, and indeed its idea of itself. The Brexit context makes these political undercurrents on foreign policy matter all the more.

Foreign policy may not matter that much to most voters, but these elections matter for foreign policy. 




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What Boris Johnson’s Big Win Means for Brexit and Scotland

13 December 2019

Thomas Raines

Director, Europe Programme

Jason Naselli

Senior Digital Editor
Thomas Raines tells Jason Naselli about the impact the large Conservative majority will have on the next phase of Brexit negotiations and Scotland’s place in the United Kingdom.

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Boris Johnson speaks after the Conservatives secured a majority in the UK general election. Photo: Getty Images.

What does the UK election result mean for Brexit and forthcoming trade negotiations with the EU?

The most important thing is that it means Brexit will definitely happen. Since the referendum, we’ve had three-and-a-half years of continued uncertainty where all outcomes were still possible. We now know that Brexit will become irreversible from 31 January.

That’s the biggest thing, because I think that will have a big psychological impact on politics, both in the UK and also on the EU side. The EU has been working with a partner that has been unsure about its direction, and perhaps some had still hoped that the process might still be reversed, but that direction is now completely clear.

Obviously the first order of business is to pass the withdrawal agreement, which should be pretty straightforward given the majority that the Conservatives have. That’s a formality now.

Then, the question becomes about the level of ambition for the next year. It is an exceptionally ambitious timetable to negotiate, ratify and implement a new relationship before the end of the transition period in December 2020.

What is achievable by the end of next year?

I think there are three possible outcomes here. One: that timetable doesn’t work and Boris Johnson follows through on his pledge to leave the transition period anyway, leading to a ‘no trade deal’ outcome.

Two: the negotiations are able to deliver something by the end of 2020, either because the depth and ambition of any agreement is relatively low level (what Michel Barnier has called a ‘vital minimum’)  and/or because they come up with some type of compromise on the process which is not called an extension, but something else: a type of temporary agreement or a new implementation period.

This is a situation where you might have a bare-bones agreement for the end of the transition period, but with an extended period of negotiation for different unresolved issues. The EU will probably insist upon level playing field guarantees and fishing access rights as a component of any such agreement.

Three: Boris Johnson breaks his manifesto pledge not to extend the transition. Now, he has stared down the barrel of leaving with no deal before and he made a political judgment that it was better to make significant compromises on his negotiating position than to follow through with ‘no deal’. I suspect he might make that same judgment again.

No option is ideal. The first is the most economically disruptive, the second means the EU will be in an even stronger position to dictate terms and the third means breaking a manifesto pledge.

How important is that end of transition deadline now? It was an important issue for the Brexit Party and hardliners in the European Research Group of Conservative MPs. But given the size of a majority, he may need to worry less about them. Is the transition deadline that important to people who voted Conservative, especially if he can show that he has taken the UK out of the EU by the end of January?

I think there may indeed be some political space for Johnson here, given the size of his majority and given that the first phase of Brexit will have been done, along with the debate about withdrawal.

There will be a lot of difficult, technical negotiations in all sorts of areas, some of which I think will become quite rancorous, but won’t necessarily become front page news in the way some of the first phase of negotiations has, not least because you won’t have the theatre of a hung parliament.

Hopefully, there will be more focus on the substance of the agreement, and the debate will be about the consequences of divergence versus staying more aligned with the EU, which is basically the central question now about the future relationship.

I still think for UK prime ministers to pick arbitrary dates, and then to make domestic political promises based around them, actually undermines the UK’s negotiating position. It would be in Britain’s interest to have more flexibility rather than a ticking clock.

Moving to the other big story from the night, the SNP won 48 of 59 seats in Scotland. How does the debate over Scotland’s future in the United Kingdom play out from here?

The SNP has really strengthened its position, more than many expected. This is now set up for a huge constitutional struggle over the future of the United Kingdom.

I think there is a key dilemma for Scottish independence supporters, which is that on the one hand Brexit greatly strengthens the political case for independence. The difference between the political preferences in Scotland and the rest of the UK, particularly in England, is a perfect demonstration of that.

At the same time, once the UK has left the EU, independence becomes much more difficult technically and economically. There will be many of the same difficulties that there have been in discussing Northern Ireland’s relationship with the Republic of Ireland. There will be a difficult debate over the currency. There are all sorts of challenges to creating a trade or regulatory border between England and Scotland. This is particularly true if there is a harder Brexit outcome, where Britain leaves the EU without a large amount of regulatory alignment.

On demands for a second independence referendum, I think in the first instance Boris Johnson will simply refuse to hold one. It’s probably not in his short-term interest to do anything else. Theresa May played it this way in 2017, repeatedly saying ‘now is not the time’.

In a similar way I think Johnson will just try to ride the pressure out, to the point where the SNP will need to face the challenges of advocating independence with the UK outside the EU. The next flashpoint will be the elections to the Scottish Parliament in 2021.

Ultimately, though, it will become a democratically unsustainable position if Scotland continues to vote for the SNP, and refusing to sanction a second independence referendum might only reinforce that sentiment.

Follow Chatham House Twitter for more election coverage




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In Next Round of EU Negotiations, Britain Faces Familiar Pitfalls

31 January 2020

Thomas Raines

Director, Europe Programme

Professor Richard G Whitman

Associate Fellow, Europe Programme
Despite being free of the constraints and the theatre of a hung parliament, there is a risk that over the coming year the British government repeats too many of the mistakes of the withdrawal negotiations.

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The Elizabeth Tower remains under renovation on 31 January 2020. Photo: Getty Images.

Whether feared or longed for, Brexit day has arrived. It is positive for all sides that the process is thus far managed and ordered, with debts paid, rights guaranteed and borders still invisible on the island of Ireland. But in a difficult new phase of negotiations, as the UK and EU try to hammer out the terms of their relationship after 2020, Britain is at risk of repeating many of its mistakes from the withdrawal talks.

First, the government, through the negotiation timeline, has reduced its own room for manoeuvre. The failure of the initial withdrawal agreement and subsequent turbulent politics have reduced a planned 21-month transition to an 11-month one. Even though half the original negotiation time has been lost, 31 December 2020 remains in place and indeed has been written into UK law as the date the transition arrangement ends. Boris Johnson has followed Theresa May in investing symbolism and significance in an arbitrary date.

By promising not to extend negotiations, the UK is boxing itself in, creating domestic political barriers where it may well need flexibility. The familiar face of Michel Barnier, who proved adept in leading the withdrawal negotiations for the EU side, will be back in phase two to tell Britain the clock is ticking. This new timeline is intended to focus minds but more likely it will limit ambitions. 

Second, this government has continued the pattern of its predecessor in making no effort to manage public expectations about the consequences of Brexit. It is naïve to have followed the last years of British politics and expect an outbreak of sobriety and levelheadedness. The entrenched positions of each side have offered little political space or electoral incentive for realism.

During the 2020 transition period, the UK will lose the political rights of EU membership but it will retain the benefits and obligations. Most citizens and business will not be able to tell the difference. But a reckoning is inevitable. There will come a moment when the effects of this slow-motion political revolution – particularly in the hard form envisioned by Boris Johnson – become real, when the trade-offs and compromises, especially for business and the economy, will bite. The public deserve some realism about the price of sovereignty.

Third, there is a risk that government remains underprepared. While its headline goals are clear – at least in terms of what it does not want – the UK government will need thorough, realistic and coherent proposals on what it wants in every area of negotiations, and crucially develop a process by which to make political trade-offs between the demands of different sectors and issues. The government must also then prepare for their implementation in every area. This would be a huge challenge even if the final destination was already known, which it is not. 

Fourth, the continued uncertainty in the process means businesses and civil servants will again be left with little time to adapt to what will face them in January 2021 and must prepare for multiple outcomes.

‘Transition’ has always been a misleading term, since it implies clarity about the destination to which the UK–EU relationship will be transitioning. The government’s red lines for that future relationship provide a sketch: outside of the single market and the jurisdiction of the European Court of Justice, with an independent trade policy and free movement ended.

But businesses and civil servants are not likely to know until very late in the process if the basis for future trade with the EU will be in the form of a free trade agreement, to be negotiated and implemented by the end of the year, or no trade deal at all. This last outcome is a realistic prospect.

Michel Barnier speaks in the European Parliament on 29 January. Photo: Getty Images.

During withdrawal negotiations, the extensions were both unlimited in number and required decisions only at the last moment. In this phase, the talks may only be extended once, and that decision must be taken six months from the final deadline. It is difficult to see circumstances in which Boris Johnson agrees to break a political promise and manifesto pledge when he still has six more months to achieve his desired outcome.

The UK, it is often noted, is already fully compliant with EU law and this shared starting point is often cited as a reason this negotiation will be simple, since the parties begin in alignment. But this novel negotiation will create new trade barriers in goods and services rather than remove them. Trade deals are often politically difficult since they create winners and losers. The Brexit negotiations, in terms of UK–EU trade at least, will generally create only different levels of losers, on both sides of the Channel.

That means difficult politics, challenging negotiations and hard compromises, another reason to expect some ugly politics along the way and accept that failure is a plausible outcome.

We do not yet know how Brexit will change Britain in the long term, whether a settled majority will ever come to view it as political folly or liberation, choice or inevitability. If its politically fragile union can withstand the pressures of the next few years, the UK may yet find a new stable position on the EU’s periphery and, after a period of economic adjustment, begin to address the many pressing domestic challenges which have suffered from neglect amid the all-consuming Brexit saga.

But whatever happens in this next chapter, the EU can no longer be an excuse for national problems. As the UK takes back control it also returns accountability. In the future, there will be no one else to credit or to blame.




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Germany in 2020: European and Global Priorities

Invitation Only Research Event

28 February 2020 - 8:30am to 11:00am

Chatham House | 10 St James's Square | London | SW1Y 4LE

This roundtable brings together German experts to discuss the country’s role in Europe and the world. Over the course of two sessions, Germany’s EU and wider foreign policy will be examined, with speakers sharing their views on where the country finds itself at the beginning of 2020 and what drives its current priorities across a number of areas. Participants will also compare perspectives on what a post-Merkel Germany might look like, now that the future leadership of CDU, Germany’s largest political party, is under question.

The event will comprise two separate sessions. Participants are welcome to attend either one or both.

08.30 – 09.30
Germany in the EU and the Eurozone

Speaker: Mark Schieritz, Economics Correspondent, Die Zeit
Chair: Quentin Peel, Associate Fellow, Europe Programme, Chatham House

Germany’s voice remains possibly the most important in any debate within the EU, including in those around the future of the Eurozone. The country has long been seen as the stalwart of the European economy and its government has always played a key role in driving Eurozone policy. However, most recent EU-wide attempts at reform have fallen short of what many claim needs to be done to complete the monetary union. The recently announced Eurozone budgetary instrument, for instance, remains very small and only focused on investment instead of stabilisation. The German government has been reluctant to go along with French President Emmanuel Macron and his structural reform proposals, though some other member states remain sceptical of his ideas for further integration too.

How can German attitudes towards the future of the Eurozone be explained? Is the government’s resistance to ambitious EU-wide economic reforms shared across the political spectrum in Germany? What stands in the way of further Eurozone reform when it comes to other EU member states? And will Germany’s reluctance to engage with reforms in this area, make it more difficult for the country to build coalitions when it comes to other EU policy areas?

09.45 – 11.00
German Foreign Policy in Perspective

Speakers: Joshua Webb, Programme Manager, Berlin Foreign Policy Forum and the Berlin Pulse, Koerber Stiftung
Dr Nicolai von Ondarza, Deputy Head, EU/Europe Research Division, German Institute for International and Security Affairs (SWP)
Chair: Dr Uta Staiger, Executive Director, UCL European Institute

Historically, Germany has been reluctant to play too active a role on the global stage, relying on its place at the heart of Europe and the transatlantic alliance. However, the current uncertain global context appears to have led to some rethinking on how the country can ensure its voice is being heard internationally, especially where its values are being challenged and its interests are at stake.

What drives German foreign policy in 2020? What are domestic priorities when it comes to trade, security and Germany’s place in the world? What shifts in public opinion may have been engendered by Brexit and Donald Trump’s presidency? What does the rise of China – and China’s growing interest in Europe – mean for Germany’s wider Asia policy?  Finally, what role will Germany play in a post-Brexit Europe? And what are the country’s priorities in its future relationship with the UK?

The speakers will discuss these and other questions, sharing the findings of a recent German public opinion survey and compare these with international expert perspectives. 

Event attributes

Chatham House Rule

Alina Lyadova

Europe Programme Coordinator




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Can the UK Strike a Balance Between Openness and Control?

2 March 2020

Hans Kundnani

Senior Research Fellow, Europe Programme
Rather than fetishizing free trade, Britain should aim to be a model for a wider recalibration of sustainable globalization.

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Boris Johnson speaks at the Old Naval College in Greenwich on 3 February. Photo: Getty Images.

This week the UK will start negotiating its future relationship with the European Union. The government is trying to convince the EU that it is serious about its red lines and is prepared to walk away from negotiations if the UK’s ‘regulatory freedom’ is not accepted – a no-deal scenario that would result in tariffs between the EU and the UK. Yet at the same time the story it is telling the world is that Britain is ‘re-emerging after decades of hibernation as a campaigner for global free trade’, as Boris Johnson put it in his speech in Greenwich a few weeks ago.

The EU is understandably confused. It’s a bit odd to claim to be campaigning for free trade at the exact moment you are creating new barriers to trade. If Britain were so committed to frictionless trade, it wouldn’t have left the EU in the first place – and having decided to leave, it would have sought to maintain a close economic relationship with the EU, like that of Norway, rather than seek a basic trade deal like Canada’s. 

As well as creating confusion, the narrative also absurdly idealizes free trade. Johnson invoked Richard Cobden and the idea that free trade is ‘God’s diplomacy – the only certain way of uniting people in the bonds of peace since the more freely goods cross borders the less likely it is that troops will ever cross borders’. But the idea that free trade prevents war was shattered by the outbreak of the First World War, which brought to an end the first era of globalization.

We also know that the domestic effects of free trade are more complex and problematic than Johnson suggested. Economic liberalization increases efficiency by removing friction but also creates disruption and has huge distributional consequences – that is, it creates winners and losers. In a democracy, these consequences need to be mitigated.

In any case, the world today is not the same as the one in which Cobden lived. Tariffs are at a historically low level – and many non-tariff barriers have also been removed. In other words, most of the possible gains from trade liberalization have already been realized. Johnson talked about the dangers of a new wave of protectionism. But as the economist Dani Rodrik has argued, the big problem in the global economy is no longer a lack of openness, it is a lack of democratic legitimacy.

The UK should therefore abandon this confusing and misleading narrative and own the way it is actually creating new barriers to trade – and do a better job of explaining the legitimate reasons for doing so. Instead of simplistically talking up free trade, we should be talking about the need to balance openness and economic efficiency with democracy and a sense of control, which is ultimately what Brexit was all about. Instead of claiming to be a ‘catalyst for free trade’, as Johnson put it, the UK should be talking about how it is trying to recalibrate globalization and, in doing so, make it sustainable.

In the three decades after the end of the Cold War, globalization got out of control as barriers to the movement of capital and goods were progressively removed – what Rodrik called ‘hyper-globalization’ to distinguish it from the earlier, more moderate phase of globalization. This kind of deep integration necessitated the development of a system of rules, which have constrained the ability of states to pursue the kind of economic policy, particularly industrial policy, they want, and therefore undermined democracy.

Hyper-globalization created a sense that ‘the nation state has fundamentally lost control of its destiny, surrendering to anonymous global forces’, as the economist Barry Eichengreen put it. Throughout the West, countries are all struggling with the same dilemma – how to reconcile openness and deep integration on the one hand, and democracy, sovereignty and a sense of control on the other.

Within the EU, however, economic integration and the abolition of barriers to the movement of capital and goods went further than in the rest of the world – and the evolution of the principle of freedom of movement after the Maastricht Treaty meant that barriers to the internal movement of people were also eliminated as the EU was enlarged. What happened within the EU might be thought of as ‘hyper-regionalization’ – an extreme example, in a regional context, of a global trend.

EU member states have lost control to an even greater extent than other nation states – albeit to anonymous regional rather than global forces – and this loss of control was felt intensely within the EU. It is therefore logical that this led to an increase in Euroscepticism. Whereas the left wants to restore some barriers to the movement of capital and goods, the right wants to restore barriers to the movement of people.

However, having left the EU, the UK is uniquely well placed to find a new equilibrium. The UK has an ideological commitment to free trade that goes back to the movement to abolish the Corn Laws in the 1840s – which Johnson’s speech expressed. It is difficult to imagine the UK becoming protectionist in any meaningful sense. But at the same time, it has a well-developed sense of national and popular sovereignty, and the sense that the two go together – which is why it was so sensitive to the erosion of them through the EU. This means that Britain is unlikely to go to one extreme or the other.

In other words, the UK may be the ideal country to find a new balance between openness and integration on the one hand, and a sense of control on the other. If it can find this balance – if it can make Brexit work – the UK could be a model for a wider recalibration of sustainable globalization. That, rather than fetishizing free trade, is the real contribution the UK can make.

A version of this article was originally published in the Observer.




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Virtual Breakfast: Europe in the Age of COVID-19: Priorities and Debates

Invitation Only Research Event

6 May 2020 - 9:00am to 10:00am

Event participants

Duncan Robinson, Charlemagne Columnist; Brussels Bureau Chief, the Economist
Chair: Pepijn Bergsen, Research Fellow, Europe Programme, Chatham House

The new European Commission had a bold new agenda when it began its work in December 2019, with climate change, digital transformation and strengthening European democracy among its priorities. Less than six months later, the European continent is in the midst of the worst crisis since the second World War and business as usual has been taken over by crisis management.

Has COVID-19 monopolized the agenda in Brussels? What priorities are still on the table and what debates have fallen victim to the coronavirus? Is the current crisis reigniting and exacerbating existing faultlines in the EU or creating new ones?

Reflecting on his first four months as the Economist’s Charlemagne columnist, the speaker will share what decision-making in Brussels looks like during a pandemic and what debates are dominating conversations in the EU capital today.

Event attributes

Chatham House Rule

Alina Lyadova

Europe Programme Coordinator




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The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation [Protein Structure and Folding]

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.




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An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1 [Enzymology]

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.




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Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena [Enzymology]

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.




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Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V [Glycobiology and Extracellular Matrices]

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.




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The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase [Glycobiology and Extracellular Matrices]

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.




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NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling [Microbiology]

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.




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Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair [Enzymology]

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.




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It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly [RNA]

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.