The Mre11/Rad50/NBS1 (MRN) protein complex and ATMIN protein mediate ATM kinase signaling in response to ionizing radiation (IR) and chromatin changes, respectively. NBS1 and ATMIN directly compete ...for ATM binding, but the molecular mechanism favoring either NBS1 or ATMIN in response to specific stimuli is enigmatic. Here, we identify the E3 ubiquitin ligase UBR5 as a key component of ATM activation in response to IR. UBR5 interacts with ATMIN and catalyzes ubiquitination of ATMIN at lysine 238 in an IR-stimulated manner, which decreases ATMIN interaction with ATM and promotes MRN-mediated signaling. We show that UBR5 deficiency, or mutation of ATMIN lysine 238, prevents ATMIN dissociation from ATM and inhibits ATM and NBS1 foci formation after IR, thereby impairing checkpoint activation and increasing radiosensitivity. Thus, UBR5-mediated ATMIN ubiquitination is a vital event for ATM pathway selection and activation in response to DNA damage.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation. All RNA interference pathways use small single-stranded RNA ...(ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5'-phosphorylated DNA guides, 13-25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5'-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through ...high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
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•Sequences flanking the Par3 CR3 consensus PKC site cooperate to inhibit aPKC•A Par3 CR3 inhibitory arm disrupts aPKC P-loop/αB/αC contacts and αC-helix position•Mutating either CR3 arm switches Par3 into an efficient aPKC substrate in vitro•Equivalent Bazooka substitutions alter its apical localization to AJs in vivo
Par3 is required for aPKC membrane recruitment, yet it polarizes to adherens junctions upon phosphorylation. Soriano et al. show that Par3 antagonizes active aPKC kinase by separating crucial N-lobe contacts. Disrupting high-affinity Par3 contacts switches it to an efficient aPKC substrate and polarizes Par3/Bazooka from apical domains to adherens junctions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment ...during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance ...function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing.
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•The histone methyltransferase SETDB1 is essential for male mouse meiosis•The meiotic DDR network recruits SETDB1 to the XY pair, where it induces H3K9me3•SETDB1 deletion perturbs meiotic sex chromosome remodeling and silencing•SETDB1 ensures timely expression of meiotic and post-meiotic genes
During male meiosis in mammals, the asynapsed regions of the X and Y chromosomes retain DNA double-strand breaks (DSBs), which triggers silencing of the sex chromosomes, a process essential for fertility. Hirota et al. show that meiotic DSB factors recruit the H3K9-methyltransferase SETDB1, inducing XY-chromatin remodeling and silencing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde ...transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The REF/ALY mRNA export adaptor binds TAP/NXF1 via an arginine-rich region, which overlaps with its RNA-binding domain. When TAP binds a REF:RNA complex, it triggers transfer of the RNA from REF to ...TAP. Here, we have examined the effects of arginine methylation on the activities of the REF protein in mRNA export. We have mapped the arginine methylation sites of REF using mass spectrometry and find that several arginines within the TAP and RNA binding domains are methylated in vivo. However, arginine methylation has no effect on the REF:TAP interaction. Instead, arginine methylation reduces the RNA-binding activity of REF in vitro and in vivo. The reduced RNA-binding activity of REF in its methylated state is essential for efficient displacement of RNA from REF by TAP in vivo. Therefore, arginine methylation fine-tunes the RNA-binding activity of REF such that the RNA-protein interaction can be readily disrupted by export factors further down the pathway.
Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A ...reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies
. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response
or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy
-reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
YAP1 gene fusions have been observed in a subset of paediatric ependymomas. Here we show that, ectopic expression of active nuclear YAP1 (nlsYAP5SA) in ventricular zone neural progenitor cells using ...conditionally-induced NEX/NeuroD6-Cre is sufficient to drive brain tumour formation in mice. Neuronal differentiation is inhibited in the hippocampus. Deletion of YAP1's negative regulators LATS1 and LATS2 kinases in NEX-Cre lineage in double conditional knockout mice also generates similar tumours, which are rescued by deletion of YAP1 and its paralog TAZ. YAP1/TAZ-induced mouse tumours display molecular and ultrastructural characteristics of human ependymoma. RNA sequencing and quantitative proteomics of mouse tumours demonstrate similarities to YAP1-fusion induced supratentorial ependymoma. Finally, we find that transcriptional cofactor HOPX is upregulated in mouse models and in human YAP1-fusion induced ependymoma, supporting their similarity. Our results show that uncontrolled YAP1/TAZ activity in neuronal precursor cells leads to ependymoma-like tumours in mice.
Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a ...sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.
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•Expression of divergent noncoding RNAs is repressed by Rap1•Rap1 prevents initiation of divergent noncoding transcription near its binding sites•Rap1 provides directionality toward productive transcription
Wu et al. characterize how divergent noncoding RNAs are repressed at highly expressed gene promoters in yeast. The authors identified that the sequence-specific transcription factor Rap1 restricts transcription initiation in the divergent direction locally near its binding site, thereby controlling promoter directionality.
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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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