In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key ...antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.
Synopsis
RNA interference (RNAi) is inefficient as an antiviral mechanism in mammals in part because it is suppressed by the type I interferon (IFN) pathway. This suppression can be mediated by type I IFN induced RIG‐I‐like helicase LGP2. LGP2 inhibits Dicer‐dependent processing of dsRNA, a prerequisite for RNAi. LGP2 inhibition of Dicer may have evolved to preserve dsRNA for stimulation of interferon‐dependent immunity.
A proteomics approach identifies LGP2 as a Dicer interactor.
LGP2 interacts with Dicer to prevent cleavage of dsRNA into small interfering RNAs in vitro and in cells.
LGP2 expression blocks dsRNA‐mediated RNAi (dsRNAi) in differentiated cells that lack a competent IFN system.
Genetic ablation of LGP2 uncovers dsRNAi in some cells.
Dicer inhibition by LGP2 may help to preserve viral dsRNA as substrate for innate immune receptor signalling, reinforcing the dominance of type I interferon signalling over RNAi in vertebrate antiviral defences.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can ...assemble into a lasso-like t-loop configuration
, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks
. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense ...tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. We show that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. We identify Skp/Cullin/F-box(Slimb/β-transducin repeats-containing protein) (SCF(Slimb/β-TrCP)) as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Continuous cancer growth is driven by subsets of self-renewing malignant cells. Targeting of uncontrolled self-renewal through inhibition of stem cell-related signaling pathways has proven ...challenging. Here, we show that cancer cells can be selectively deprived of self-renewal ability by interfering with their epigenetic state. Re-expression of histone H1.0, a tumor-suppressive factor that inhibits cancer cell self-renewal in many cancer types, can be broadly induced by the clinically well-tolerated compound Quisinostat. Through H1.0, Quisinostat inhibits cancer cell self-renewal and halts tumor maintenance without affecting normal stem cell function. Quisinostat also hinders expansion of cells surviving targeted therapy, independently of the cancer types and the resistance mechanism, and inhibits disease relapse in mouse models of lung cancer. Our results identify H1.0 as a major mediator of Quisinostat's antitumor effect and suggest that sequential administration of targeted therapy and Quisinostat may be a broadly applicable strategy to induce a prolonged response in patients.
Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 ...kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK‐dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK‐dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation‐independent accumulation of ULK substrates and kinase activity‐regulated recruitment of autophagy proteins to ubiquitin‐positive structures.
SYNOPSIS
Serine/threonine kinase ULK is the sole protein kinase in the autophagic signalling cascade and a key autophagy regulator. This study identifies the VPS34 complex component VPS15 as a novel ULK substrate in mammalian cells, and reveals a role for ULK‐VPS15 signalling in autophagosome formation.
Unbiased phosphoproteomics and in vitro kinase assays identify several novel ULK substrates.
ULK phosphorylates VPS15 and PRKAG2, components of VPS34 and AMPK complexes, respectively.
The ULK‐VPS15 signalling axis regulates VPS34 complex activity in vitro, and autophagy in cells.
VPS15 knockout cells accumulate subsets of ULK phospho‐substrates.
Upon VPS15 loss, autophagy proteins redistribute to ubiquitin‐positive condensates in an ULK‐ and VPS34‐dependent manner.
ULK‐dependent phosphorylation of the VPS34 complex component VPS15 regulates starvation‐induced autophagy in mammalian cells.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is ...activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.
Synopsis
Mitotic phosphorylation by CDK and Polo kinases is reversed at the exit from mitosis. Phosphoproteome dynamics show ordering of dephosphorylation through successive inactivation of these kinases, but also significant new phosphorylation through late mitotic kinases.
SILAC proteomics in synchronized yeast defines phosphorylation dynamics with high temporal resolution.
Similar numbers of phosphorylation and dephosphorylation events take place during mitotic exit.
Kinase activation and inactivation orchestrates phosphorylation and dephosphorylation timings.
A full CDK consensus motif provides the most selective means of cell cycle regulation.
Ordered target dephosphorylation upon successive inactivation of CDK and Polo kinases is unexpectedly paralleled by a similarly large number of new phosphorylation events via late mitotic kinases.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Mammalian telomeres protect chromosome ends from aberrant DNA repair
. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the ...terminal telomere repeat sequence within a lariat T-loop structure
. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability
. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: ...initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.
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•GABARAP binds directly to the centriolar satellite protein PCM1 through a LIR motif•GABARAP-PCM1-positive centriolar satellites are found at early-stage autophagosomes•PCM1 regulates GABARAP-specific autophagosome formation and GABARAP degradation•The centriolar satellite E3 ligase Mib1 drives ubiquitination of GABARAP
Joachim et al. show PCM1-positive centriolar satellites regulate the formation of GABARAP-positive autophagosomes. GABARAP stability is regulated by PCM1, most likely through Mib1-driven ubiquitination. This study reveals new insights into the poorly understood communication between the centrosome and autophagosomes during starvation-induced autophagy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core ...complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.
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•FANCB, FANCL, and FAAP100 form a symmetric dimer of trimers•FANCL is ideally poised for the symmetric mono-ubiquitination of FANCI-FANCD2•Two separate FANCC-FANCE-FANCF complexes bind to the opposing poles of FANCB-FANCL-FAAP100•FANCC-FANCE-FANCF stabilizes FANCI-FANCD2 for efficient mono-ubiquitination
Mono-ubiquitination of FANCI-FANCD2 by the Fanconi anemia core complex activates a major DNA interstrand-crosslink repair pathway important for genome stability maintenance. Here, Swuec et al. reveal the structural basis of this reaction by showing that the core complex exists as a dimeric catalytic module for the symmetric mono-ubiquitination of FANCI-FANCD2.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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