22 July 2020

27 July 2020

10 August 2020

11 August 2020

20 August 2020

26 August 2020

16 September 2020

The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75–Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Will Africans’ calls for better democracy be met? The World Today mhiggins.drupal 29 July 2022

Voters want the continent’s ageing leaders to step aside to usher in a new age of political engagement and robust democracy, say the experts of Afrobarometer.

Across Africa, recent years have been marked by both encouraging democratic highs and troubling anti-democratic lows. Notable advances from last year include the Gambia’s successful presidential election, a ruling-party transition in Zambia and the first democratic transfer of power in Niger. In the lead up to this, add Malawi’s retake of its flawed presidential election in 2020 and an earlier succession of oustings of long-serving autocrats in Sudan, Zimbabwe and the Gambia. 

Contrast these gains, though, with setbacks elsewhere, including increasing restrictions on opposition parties in Benin, Senegal and Tanzania; the use of violence and intimidation during elections in Côte d’Ivoire and Uganda; and military coups, with the latest in Burkina Faso this year and last year in Chad, Mali, Sudan and Guinea.

These contradictory developments join dire warnings from experts that democracy is losing ground on the continent. But what can we learn about the state of democracy on the continent from Africans themselves?

Afrobarometer, a pan-African, non-partisan research network, has been surveying people about their views on democracy, governance and quality of life for more than 20 years. After interviewing nearly 50,000 citizens across 34 countries during Afrobarometer Round 8, which spans 2019-2021, we find that despite the efforts of some leaders to undermine democratic norms, Africans remain committed to democracy and democratic institutions.

They believe that the military should stay out of politics, that political parties should freely compete for power, that elections are an imperfect but essential tool for choosing their leaders, and that it is time for the old men who cling to power to step aside.

But their political reality falls short of these expectations. The perception of widespread and worsening corruption is particularly corrosive, leaving people increasingly dissatisfied with political systems that are yet to deliver on their aspirations to live in societies that are democratically and accountably governed. And although citizens find myriad ways to voice their concerns, they feel that their governments are not listening.

Simply put, Africans want more democratic and accountable governance than they think they are getting.

Africans’ democratic aspirations

Over the past decade, democracy watchers have been alarmed by declining trends in Africa. Concerns have been exacerbated in the past two years as some governments have taken advantage of the Covid pandemic to limit freedoms, restrict fair campaigning or postpone elections. Activists fear that supposedly temporary rollbacks in hard-won governance reforms could become permanent.

But for the most part, African citizens remain committed to democracy and democratic institutions. Across 30 countries that Afrobarometer has surveyed consistently since Round 5 (2011–2013), most indicators are strong and quite steady.

For example, seven in 10 Africans say that ‘democracy is preferable to any other kind of government’. While this is down modestly from 73 per cent a decade ago, more specific indicators seem to affirm popular commitment to democracy. Large and steady majorities consistently reject authoritarian alternatives, including one-person or ‘strongman’ rule (82 per cent), one-party rule (77 per cent) and military rule (75 per cent), which is clearly rejected even in many of the countries rocked by recent military coups.

Africans also express strong support for a limit to presidential terms, a feature of democratic governance that researchers and activists argue nurtures political participation, demonstrates that change via the ballot box is possible, and reduces the risk of personality cults, authoritarianism, corruption and coups. Across 34 countries, an average of 76 per cent favour limiting their presidents to two terms, including a majority (54 per cent) who ‘strongly’ support this rule. Term limits enjoy majority support in every surveyed country. 

The public’s democratic commitment is undergirded by strong and in some cases growing support for core democratic institutions. Support for multiparty competition and parliamentary oversight of leaders remains steady, while expectations that governments should be accountable to the courts have increased significantly over the past decade.

In addition, growing numbers of people say it is more important to have a government that is accountable to the people rather than one that just ‘gets things done’, an especially strong indicator of deepening commitment to democratic norms among citizens. 

Trouble at the polling booth

Elections remain a central, though controversial, institution of democracy for Africans. They have served as the foundation for real change, as in Zambia last year. But in other cases, such as Uganda’s January 2021 poll, they have been marred by violence and human rights abuses, as well as the weaponization of Covid to justify restrictions on campaigning.

The public is also sceptical about the capacity of elections to bring about real change: fully 50 per cent say they do not think elections are effective in enabling voters ‘to remove from office leaders who do not do what the people want’.

At the same time, large majorities report positively on their country’s election environment. Asked about their most recent election, at least eight in 10 say they did not observe intimidation (87 per cent) or interference (81 per cent) by security forces and did not fear violence (80 per cent).

We must keep in mind that these encouraging averages can obscure deep problems in some countries. For example, while only 3 per cent of Namibians say votes are ‘often’ not counted fairly, between a quarter and one-third cite inaccurate counts as a frequent problem in Zimbabwe, Sudan and Gabon. 

In addition, confidence in the fairness of the media environment is drastically lower, on average just 36 per cent.

But perhaps most importantly, almost nine in 10 Africans (87 per cent) say they are free to vote as they choose, including sizeable majorities in every surveyed country. And a solid majority of 63 per cent rate their most recent election as completely or mostly free and fair. 

All of this may help to explain still-strong support for competitive elections as the best system for selecting leaders. A robust three-quarters confirm their commitment to elections, though this has fallen slightly over the past decade, probably reflecting disillusionment with electoral processes that are too often torn by violence and produce contested results. 

A growing number of people may also be recognizing that elections, especially poor-quality ones, are not enough to guarantee democracy and better governance, and that a healthy democracy must include such other features as government accountability, respect for the rule of law, responsiveness and citizen participation.

The ‘democratic disappointment’ gap

To what extent does political reality align with Africans’ democratic aspirations? Our findings suggest that it is falling well short of expectations.

While a slim majority has steadily reported that their country is a ‘full democracy’ or one ‘with minor problems’ over the past decade, satisfaction, however, has dropped to 43 per cent in that time. 

What explains this growing dissatisfaction? Other indicators of democratic supply offer some clues. While ratings of election quality have held steady, favourable public assessments of presidential accountability to parliament and to the courts have both declined.

The rising scourge of corruption

But one of the most significant driving factors may be burgeoning corruption, a trend that appears to parallel declining democratic satisfaction. On average across 34 countries, around six in 10 say both that corruption in their country increased over the past year, and that their government is doing a poor job of controlling it.

These perceptions matter. Over time, when perceptions of corruption rise or fall, levels of dissatisfaction with democracy tend to follow suit. 

In South Africa, dissatisfaction with democracy grew steadily alongside scandals involving President Jacob Zuma, and has continued to rise under his successor, Cyril Ramaphosa, whose office has been tainted by ‘Farmgate’ and a major Covid-relief scandal. The ‘Fishrot’ scandal in Namibia has had similar consequences.

Are governments listening?

African citizens are raising their voices, calling on their governments to fulfil their democratic aspirations. Since April 2017, the Carnegie Endowment for International Peace has recorded more than 70 episodes in 35 African countries of protests focused on issues ranging from demands for democracy in eSwatini to resisting police brutality, presidential third-term attempts and Covid restrictions. 

Citizen participation and government responsiveness are cornerstones of democracy. But are governments listening?

Voting is the most obvious and popular way for citizens to express themselves, and Africans take advantage of this opportunity. Two-thirds said they voted in their most recent national election. But elections occur only occasionally, and they force individuals to compress a wide array of views into very few choices. How do Africans find their voice during the long intervals between elections?

Many invest in personal efforts to act as agents of change. In fact, nearly half say they joined with others to raise an issue at least once in the past year, and a third contacted a political leader. A quarter report they acted with others to request government action. Less common but still important modes of engagement include asking for help from or lodging a complaint with government, contacting the media, and joining a demonstration.

These robust levels of citizen engagement suggest that people feel they can make a difference. Unfortunately, decision-makers aren’t always receptive or responsive to citizen voices. Less than a quarter of people think local government officials listen to them – and even fewer think their members of parliament do. 

What is more troubling is that fully two-thirds say they are at risk of retaliation or some form of negative consequences if they take action by reporting incidents of corruption. 

Lack of government responsiveness and respect for popular voices may have direct implications for both citizen engagement and citizen satisfaction. For example, we find that people are more likely to contact leaders or take other actions to solve problems if they believe that government officials respect and listen to them; that they will get a response if they raise an issue; and if they do not need to fear retaliation. 

Similarly, when we compare country averages for government responsiveness to the percentage of citizens who are satisfied with democracy, we again find positive associations. 

When governments are responsive, citizens are more likely to engage in addressing community needs and to be satisfied with their political system and optimistic about the future. Respectful and responsive governance has the potential to spur citizen action to solve critical development challenges – and may be the cure for what ails democracy.

Eight ways to build better African cities The World Today mhiggins.drupal 2 August 2022

Young professionals from across the continent tell Emmanuel Adegboye how city life could be improved: from high-speed rail to people-centred urban planning.

Ahmed Elsawy, 33, Director of Talent
Cairo, Egypt
We must amend employment law in Egypt to support individual contractors to match with the global demand for short-term and project-based assignments within the tech and service industries. While we have a new, decent education system, I think we should care more about foreign languages to have a higher rank when it comes to the global competition of skilled workers.

Iman Abubaker, 31, Urban Mobility Project Manager, WRI Africa
Addis Ababa, Ethiopia
Rapid urbanization and increased motorization have exacerbated the city’s urban challenges. Addis Ababa would benefit from safer street design and people-centred city planning. Urban amenities should be located within walking and cycling distances. For longer trips, the city needs to invest in improving the accessibility, safety, integration and multimodality of its public transport system. I would love to see more pockets of green spaces and parks all around the city. 

Bree, 31, Project Manager
Nairobi, Kenya
I have a love-hate relationship with Nairobi. I spent four years smack in the middle of the city while attending the University of Nairobi. Being in the middle of all the hustle and bustle made the transition to a sleepy-ish coastal town easy. I would happily trade matatus [shared taxis] for tuktuks any day. I do miss the conveniences that come with a big city like a 24-hour grocery store and delivery services on those lazy days.

Mfon Bassey, 30, Co-Founder, TalentX Africa
Lagos, Nigeria
The government should improve the road networks and public transport systems, because the common challenge most Lagosians face is commuting from point A to B without traffic. There are so many private cars on the road because the public transport system isn’t optimally efficient yet. Once you take away the commute time most workers spend just to get work done, we’ll surely have happier Lagosians.

Olga Kiconco, 32, Innovation Strategist
Kampala, Uganda
As one of Africa’s fastest-growing cities with a projected 112 per cent population growth by 2035, there are a number of critical changes that need to be made in preparation for this. Our leaders should embrace coherent policies that will catalyze socio-economic transformation. We need to hold them accountable for better infrastructure and delivery of public services, while taking personal responsibility to protect our environment against the prevalent threat of climate change.

Etienne Amougou, 30, Curator/Arts Project Manager
Yaounde, Cameroon
What would make Yaounde better would be a good ecosystem that provides more opportunities for young people. If it was possible, I would like to see the creation of more cultural spaces, like parks, zoos, cinemas and sport areas. Also, we could use a more effective approach to waste management – sometimes we have trash everywhere in the ’hood.

Valentino Fernandez, 23, Writer
Johannesburg, South Africa
 We need better transportation to bridge the inequality gap and allow the youth to access spaces to be inspired and create change. Apartheid spatial planning is still affecting us. People of colour were relegated to the outskirts of the city, and very little has changed. It’s virtually impossible to move out of your childhood home, which means you’re looking at a two-hour commute every morning and two more hours to get home. I would like a reliable, affordable, high-speed rail system.

Jean-Louis Mbaka, 34, Co-Founder and Director, Education at Kinshasa Digital
Kinshasa, DRC
Our youth must receive a sufficient education that is in line with the strategic requirements of their future workplaces. By 2030, more than 130 million jobs in Africa will require digital skills, according to the International Finance Corporation. To close the gap between the conventional educational system and the labour market, our organisation is providing training for digital jobs. Initiatives like ours must be supported if the current and next generations are to have the means for their economic and social advancement. Scaling up investments in vital facilities like the internet is also necessary.

The Climate Briefing: The nexus of water security and climate policy Audio NCapeling 22 August 2022

Examining the crossover between water security and climate change with the next two COPs taking place in regions with a history of being water stressed.

What should policymakers and negotiators from the Middle East and Africa working on water security focus on at COP27?

What does it mean to achieve water security? What are the main barriers or challenges? How is water security relevant to climate change?

This podcast was produced in collaboration with the UK Aid-funded Knowledge, Evidence and Learning for Development (K4D) programme which facilitates the use of evidence and learning in international development policy and programming.

Africa-Japan relations and evolving multilateralism 23 November 2022 — 9:00AM TO 10:30AM Anonymous (not verified) 17 November 2022 Online

This panel discussion reflects on the outcomes of TICAD 8 in 2022 and looks forward to TICAD 9 in 2025.

The eighth edition of the Tokyo International Conference on African Development (TICAD), held in Tunisia from 27–28 August 2022, marked the second time that Japan’s now-triennial summit was hosted in an African country, after TICAD 6 was held in Kenya in 2016.

The summit was attended by 48 representatives of African countries and at least 20 heads of state and government and included a pledge by the Japanese government to commit $30 billion in public and private finance to Africa over the next three years.

In reaffirming the three pillars of TICAD 8 – revolving around the economy, societal resilience, and peace and stability – the newly adopted Tunis Declaration (28 August 2022) also outlined some of the key projects underpinning Japan’s pledge, including a $4 billion fund for a Green Growth Initiative with Africa (GGA).

2023 will mark 30 years since the inception of TICAD in 1993 and ten years since the African Union (AU)’s adoption of its flagship Agenda 2063, on which the Tunis Declaration placed distinct emphasis.

This panel discussion reflects on the outcomes of TICAD 8 in 2022 and looks forward to TICAD 9 in 2025, exploring wider developments in summitry, Africa-Asia relations, and modes of multilateralism.

Questions explored include:

• How has international summitry evolved over the past three decades since the inception of TICAD in 1993, which represented the first periodic high-level summit engagement with Africa by a ‘non-traditional’ partner?
• Looking ahead to TICAD 9 in 2025, what are the priorities for enforcing the stated tenets of TICAD – ‘African ownership, international partnership, inclusivity and openness’ – in cooperation efforts?
• What lessons can be drawn from TICAD’s co-partnership approach (with the African Union Commission and others) – particularly given increasing calls for AU membership of the G20 and Prime Minister Kishida’s pledge at TICAD 8 to support a permanent African UNSC seat during its non-permanent membership in 2023–24? Beyond membership, what are the priorities for furthering agency?
• How are Africa-Asia relations evolving and diverging? How are Japan and other Asian countries perceived by different African countries?

This event is the third in the Chatham House – Japan House London webinar series (2022-2023). The series is held in partnership with Japan House London. You can watch previous webinars from the series here.

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Sophos X-Ops unveils five-year investigation tracking China-based groups targeting perimeter devices

Sophos X-Ops unveils five-year investigation tracking China-based groups targeting perimeter devices

Tricia Rowlison
Dec 1, 2020; 19:2090-2103
Research

Xiao-Ting Wen
Dec 17, 2020; 0:RA120.002119v1-mcp.RA120.002119
Research

Kyle Swovick
Dec 28, 2020; 0:RA120.002301v1-mcp.RA120.002301
Research

Yi-Han Lin
Dec 1, 2020; 19:2030-2046
Research

Sylwia Struk
Dec 28, 2020; 0:RA119.001766v1-mcp.RA119.001766
Research

Julia Knöckel
Dec 22, 2020; 0:RA120.002432v1-mcp.RA120.002432
Research

Nadine Prust
Dec 17, 2020; 0:RA120.002232v1-mcp.RA120.002232
Research

R. Greg Stacey
Nov 4, 2020; 0:RA120.002275v1-mcp.RA120.002275
Research

Madison T Wright
Nov 18, 2020; 0:RA120.002168v1-mcp.RA120.002168
Research

Xinting Zhang
Dec 8, 2020; 0:RA120.002306v1-mcp.RA120.002306
Research

Mingkun Yang
Nov 24, 2020; 0:RA120.002144v1-mcp.RA120.002144
Research

Ka-Won Kang
Nov 30, 2020; 0:RA120.002169v1-mcp.RA120.002169
Research

Jianhua Wang
Nov 4, 2020; 0:RA120.002375v1-mcp.RA120.002375
Research

Diana Samodova
Dec 1, 2020; 19:2139-2156
Technological Innovation and Resources

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors. By promoting GTP binding, guanine nucleotide exchange factors induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found that the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane–localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the residues that are required for the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. Our results demonstrate that the amphipathic motifs of Arfrp1 and Arl14 are sufficient for determining specific subcellular localizations in a GTP-independent manner, suggesting that the membrane association and activation of some Arf proteins are uncoupled.

The heterotrimeric G proteins are known to have a variety of downstream effectors, but Gs was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated Gs can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by Gs-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gαs subfamily regulates activity of Rho GTPases extends our understanding of Gαs activity and establishes RhoGEF coupling as a universal Gα function.

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5' end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

Melissa Gómez
Dec 1, 2020; 61:1658-1674
Research Articles

Elisa Vidal
Dec 1, 2020; 61:1733-1746
Research Articles

Daphne E.C. Boer
Dec 23, 2020; 0:jlr.RA120001043v1-jlr.RA120001043
Research Articles

Nicholas D. LeBlond
Dec 1, 2020; 61:1697-1706
Research Articles

Auguste Dargent
Dec 1, 2020; 61:1776-1783
Research Articles

Ryunosuke Ohkawa
Dec 1, 2020; 61:1577-1588
Research Articles