EMBO Journal最新文献

F-box proteins are the substrate recognition modules of the SCF (SKP1-Cullin-F-box) E3 ubiquitin ligase complex. FBXO42, an understudied member of this family, has recently emerged as a modulator of key cellular processes, including cell cycle progression, the DNA damage response, and glioma stem cell survival. In this study, we define the function of FBXO42 as a major regulator of the protein phosphatase PP4. Phosphoprotein phosphatases (PPPs) have a broad array of substrates, hence necessitating tight regulation. We observe that FBXO42 ubiquitinates the PP4 complex to govern the assembly of regulatory and catalytic subunits, with the net effect of restraining the latter's phosphatase activity. FBXO42 depletion unleashes PP4 activity, with broad cellular effects, highlighting FBXO42 as a novel regulatory node in ubiquitin-mediated signalling for future therapeutic exploitation.

ARMS (ankyrin repeat-rich membrane spanning) is a scaffold protein essential for neurotrophic signaling, synaptic development, and cytoskeletal remodeling. Despite its central role in neuronal function, how ARMS is regulated at the molecular level remains poorly understood. Here, we identify GABARAP, an Atg8-family autophagy adaptor, as a novel ARMS-binding protein that directly interacts with its N-terminal ankyrin repeats. We present the crystal structure of the ARMS-GABARAP complex, revealing an atypical interaction mode distinct from canonical LIR-dependent Atg8 interactions. Remarkably, ARMS specifically binds to the GABARAP subfamily of Atg8 proteins, setting it apart from the LC3 subfamily. Functional analysis demonstrates that GABARAP negatively regulates ARMS-mediated dendritic spine development and maturation in hippocampal neurons. Additionally, disrupting the ARMS-GABARAP complex using ankyrin-derived peptides alters ARMS subcellular localization, increasing its accumulation in the soma of neurons. Collectively, our findings uncover a novel ARMS-GABARAP interaction mechanism, establish the regulatory role of this complex in neuronal protein homeostasis, and suggest potential therapeutic strategies for targeting scaffold protein interactions in neurodevelopmental and neurodegenerative disorders.

Cell fate decisions require tight regulation of gene expression. In planarians, highly regenerative flatworms, the mRNA modification N⁶-methyladenosine (m⁶A) modulates progenitor production and fate. However, the mechanisms governing m⁶A deposition in the planarian transcriptome, and the role of their expanded family of YTHDF m⁶A reader proteins in orchestrating biological functions, remain unclear. Here, we generated the first single-nucleotide resolution map of m⁶A in planarians, and revealed that simple sequence rules guide m⁶A deposition, facilitating the flexible evolutionary gain and loss of these marks. Functional analyses of the five YTHDF planarian m⁶A readers revealed that while individual reader expression is dispensable, together, the planarian YTHDF proteins regulate the production of specific progenitor lineages and overall body size. Collectively, our findings uncover a robust, redundant regulatory architecture for cell fate control in planarians, characterized by multiple m⁶A sites per gene and coordinated m⁶A reader expression. This architecture is essential for proper lineage resolution and provides insights into the evolutionary dynamics of the m⁶A landscape.

Cellular and environmental stress triggers the rapid and global reprogramming of gene transcription by coordinated recruitment of a limited number of key inducible transcription factors to cis-regulatory elements. Here, we performed a comprehensive analysis of different stress models and observed that co-induced genes are generally located in close genomic proximity. By integrating gene expression and transcription factor binding resources across different stress models, we identify an enrichment for clusters in which only one of the clusters' promoters recruits the key transcription factors, reminiscent of Epromoters-a type of cis-regulatory element that displays both promoter and enhancer function. Epromoter-regulated clusters were frequently found regardless of the stress or inflammatory response. Predicted Epromoters displayed enhancer activity and regulated clusters of stress-response genes independently of their genomic location. These findings imply that Epromoters are central regulatory elements that control gene clusters in response to acute perturbations.

The subcellular localization of many mRNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for most of these RNAs, the mechanisms that govern their localization are unknown. Here, using subcellular fractionation and single-molecule RNA FISH, we found that loss of TDP-43 results in increased accumulation of hundreds of mRNAs in neurites. Using high-throughput functional assays in cells and high-throughput binding assays in vitro, we subsequently identified specific regions within these mRNAs that mediate their TDP-43-dependent localization and interaction with TDP-43. We found that the same regions also mediated TDP-43-dependent mRNA instability, suggesting a mechanism by which TDP-43 regulates mRNA localization. ALS-associated mutations in TDP-43 resulted in similar mRNA mislocalization phenotypes as did TDP-43 loss in mouse dorsal root ganglia and human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of mRNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

Centromere position is specified and maintained by sequence-independent epigenetic mechanisms in vertebrate cells, with the incorporation of the centromere-specific histone H3 variant CENP-A into chromatin being a key event for centromere specification. Although many models for CENP-A incorporation have been proposed, much remains unknown. In this study, we reveal that the CENP-A chaperone HJURP directly binds to the C-terminal domain of chicken CENP-C in vitro and that this interaction is essential for new CENP-A incorporation in chicken DT40 cells. While existing models have suggested that HJURP is recruited by the Mis18 complex (Mis18C), here, we propose that CENP-C and Mis18C provide dual recruitment pathways for HJURP localization to centromeres in DT40 cells. We demonstrate that both HJURP localization and new CENP-A incorporation are completely abolished in Mis18C knockout cells expressing an HJURP mutant lacking CENP-C binding ability. Furthermore, co-immunoprecipitation experiments reveal that CENP-C, HJURP and Mis18C form a tight association in the chromatin fraction. These two pathways are critical for robust CENP-A incorporation to maintain centromere position in vertebrate cells.

The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.

Multicellular organisms rely on inter-organ communication networks to maintain vital parameters within a dynamic physiological range. Macrophages are central to this homeostatic control system, sensing and responding to deviations of those parameters to sustain organismal homeostasis. Here, we demonstrate that dysregulation of iron (Fe) metabolism, imposed by the deletion of ferritin H chain (FTH) in mouse parenchymal cells, is sensed by monocyte-derived macrophages. In response, monocyte-derived macrophages support tissue function, energy metabolism, and thermoregulation via a mechanism that sustains the mitochondria of parenchymal cells. Mechanistically, FTH supports a transcriptional program promoting mitochondrial biogenesis in macrophages, involving mitochondrial transcription factor A (TFAM). Moreover, FTH sustains macrophage viability and supports intercellular mitochondrial transfer from donor parenchymal cells. In conclusion, monocyte-derived macrophages cross-regulate iron and energy metabolism to support tissue function and organismal homeostasis.

Antifungal resistance in pathogenic fungi endanger global health and food supply. Wild-type fission yeast, Schizosaccharomyces pombe, can gain resistance to insults including caffeine and antifungal compounds through reversible epimutations. Resistant epimutants exhibit ectopic histone-H3K9 methylation-dependent heterochromatin islands, repressing underlying genes. Two genes whose heterochromatin island-induced repression causes resistance encode mitochondrial proteins: LYR-domain protein Cup1 and Cox1 translation regulator Ppr4. Genetic mutations, cup1-tt and ppr4Δ, that phenocopy epimutants, cause mitochondrial dysfunction, including respiratory deficiency, poor growth on non-glucose carbon sources, and elevated reactive oxygen species. Transcriptomic analyses indicate cup1-tt and ppr4Δ cells activate Pap1 transcription factor-dependent oxidative stress response and mitonuclear retrograde pathways. Pap1 nuclear localisation and recruitment to promoters of oxidoreductase and membrane transporter genes is increased, causing increased efflux activity. cup1 and ppr4 epimutants likewise show mitochondrial dysfunction phenotypes and increased efflux, explaining how heterochromatin-island epimutations cause drug resistance. Thus, wild-type cells harness epimutations that impose mitochondrial dysfunction to bypass external insults. As mitochondrial dysfunction is linked to antifungal resistance in several fungi, similar epimutations likely contribute to development of resistance in fungal pathogens.

Small noncoding RNAs (sncRNAs) are subject to 3'-end trimming and tailing activities that impact maturation versus degradation decisions during biogenesis. To investigate the dynamics of human sncRNA 3'-end processing at a global level, we performed genome-wide 3'-end sequencing of newly transcribed and steady-state sncRNAs. This revealed widespread post-transcriptional adenylation of newly transcribed sncRNAs, which came in two distinct varieties. One is characterized by oligoadenylation, which is transient, promoted by TENT4A/4B polymerases, and most commonly observed on unstable small nucleolar RNAs that are not fully processed at their 3'-ends. The other is characterized by monoadenylation, which is broadly catalyzed by TENT2 and, in contrast to oligoadenylation, stably accumulates at the 3'-end of sncRNAs, including Polymerase-III-transcribed (Pol-III) RNAs and a subset of small nuclear RNAs. Monoadenylation inhibits Pol-III RNA post-transcriptional 3'-uridine trimming and extension and, in the case of 7SL RNAs, prevents their accumulation with nuclear La protein and promotes their biogenesis towards assembly into cytoplasmic signal recognition particles. Thus, the biogenesis of human sncRNAs involves widespread mono- or oligoadenylation with divergent impacts on sncRNA fates.