EMBO Reports最新文献

In neuronal cells, the regulation of RNA is crucial for the spatiotemporal control of gene expression, but how the correct localization, levels, and function of synaptic proteins are achieved is not well understood. In this study, we globally investigate the role of alternative 3' UTRs in regulating RNA localization in the synaptic regions of the Drosophila brain. We identify direct mRNA targets of the translational repressor Pumilio, finding that mRNAs bound by Pumilio encode proteins enriched in synaptosomes. Pumilio differentially binds to RNA isoforms of the same gene, favoring long, neuronal 3' UTRs. These longer 3' UTRs tend to remain in the neuronal soma, whereas shorter UTR isoforms localize to the synapse. In cultured pumilio mutant neurons, axon outgrowth defects are accompanied by mRNA isoform mislocalization, and proteins encoded by these Pumilio target mRNAs display excessive abundance at synaptic boutons. Our study identifies an important mechanism for the spatiotemporal regulation of protein function in neurons.

Stimulator of interferon genes (STING) is essential for innate immune pathway activation in response to pathogenic DNA. Proper activation of STING signaling requires STING translocation and phosphorylation. Here, we show that dual serine/threonine and tyrosine protein kinase (DSTYK) directly phosphorylates STING Ser366 at late endosomes to promote the activation of STING signaling. We find that TBK1 promotes STING post-Golgi trafficking via its kinase activity, thereby enabling the interaction between DSTYK and STING. We also demonstrate that DSTYK and TBK1 can both promote STING phosphorylation at late endosomes. Using an in vivo Dstyk-knockout model, we showed that mice deficient in DSTYK demonstrate reduced STING signaling activation and are more susceptible to infection with a DNA virus. Together, we reveal the previously unknown cellular function of DSTYK in phosphorylating STING and our findings provide insights into the mechanism of STING signaling activation at late endosomes.

Barth syndrome (BTHS) is a rare genetic disease caused by mutations in the TAFAZZIN gene. It is characterized by neutropenia, cardiomyopathy and skeletal myopathy. Neutropenia in BTHS is associated with life-threatening infections, yet there is little understanding of the molecular and physiological causes of this phenomenon. We combined bone marrow analysis, CRISPR/Cas9 genome editing in hematopoietic stem cells and functional characterization of circulating BTHS patient neutrophils to investigate the role of TAFAZZIN in neutrophils and their progenitors. We demonstrate a partial cell intrinsic differentiation defect, along with a dysregulated neutrophil inflammatory response in BTHS, including elevated degranulation and formation of neutrophil extracellular traps (NETs) in response to calcium flux. Developmental and functional alterations in BTHS neutrophils are underpinned by perturbations in the unfolded protein response (UPR) signaling pathway, suggesting potential therapeutic avenues for targeting BTHS neutropenia.

Cable bacteria are centimeter-long multicellular bacteria conducting electricity through periplasmic conductive fibers (PCFs). Using single-strain enrichments of the genera Electrothrix and Electronema we systematically investigate variations and similarities in morphology and electrical properties across both genera. Electrical conductivity of different PCFs spans three orders of magnitude warranting further investigations of the plasticity of their conduction machinery. Using electron microscopy and elemental analyses, we show that the two cable bacteria genera have similar cell envelopes and cell-cell junction ultrastructures. Iron, sulfur, and nickel signals are co-localized with the PCFs, indicating key functional roles of these elements. The PCFs are organized as stranded rope-like structures composed of multiple strands. Furthermore, we report lamellae-like structures formed at the cell-cell junctions with a core layer connecting to the PCFs, and intriguing vesicle-like inner membrane invaginations below the PCFs. Finally, using bioinformatic tools, we identify a cytochrome family with predicted structural homology to known multi-heme nanowire proteins from other electroactive microorganisms and suggest that these cytochromes can play a role in the extra- or intercellular electron conduction of cable bacteria.

Embryonic and epiblast stem cells in pre-and post-implantation embryos are characterized by their naïve and primed states, respectively which represent distinct phases of pluripotency. Thus, cellular transition from naïve-to-primed pluripotency recapitulates a drastic metabolic and cellular remodeling after implantation to adapt to changes in extracellular conditions. Here, we found that inhibition of AMPK occurs during naïve transition with two conventional inhibitors of the MEK1 and GSK3β pathways. The accumulation of glycogen due to iGSK3β is responsible for AMPK inhibition, which accounts for high de novo fatty acid synthesis in naïve (ESCs). The knockout of glycogen synthase 1 in naïve ESCs; GKO, resulting in a drastic glycogen loss, leads to a robust AMPK activation and lowers the level of fatty acids. GKO loses cellular characteristics of naïve ESCs and rapidly transitioned to a primed state. The characteristics of GKO are restored by the simultaneous AMPK KO. These findings suggest that high glycogen in epiblast within pre-implantation blastocyst may act as a signaling molecule for timely activation of AMPK, thus ultimately contributing to transition to post-implantation stage epiblast.

Internal translation initiation sites, as revealed by ribosome profiling experiments can potentially drive the translation of many N-terminally truncated proteoforms. We report that internal short open reading frame (sORF) within coding sequences regulate their translation. nTRIP6 represents a short nuclear proteoform of the cytoplasmic protein TRIP6. We have previously reported that nTRIP6 regulates the dynamics of skeletal muscle progenitor differentiation. Here we show that nTRIP6 is generated by translation initiation at an internal AUG after leaky scanning at the canonical TRIP6 AUG. The translation of nTRIP6 is repressed by an internal sORF immediately upstream of the nTRIP6 AUG. Consistent with this representing a more general regulatory feature, we have identified other internal sORFs which repress the translation of N-terminally truncated proteoforms. In an in vitro model of myogenic differentiation, the expression of nTRIP6 is transiently upregulated through a mechanistic Target of Rapamycin Complex 1-dependent increase in translation initiation at the internal AUG. Thus, the translation of N-terminally truncated proteoforms can be regulated independently of the canonical ORF.

Basal-like breast cancers exhibit distinct cellular heterogeneity that contributes to disease pathology. In this study we used a genetic mouse model of basal-like breast cancer driven by epithelial-specific inactivation of the Hippo pathway-regulating LATS1 and LATS2 kinases to elucidate epithelial-stromal interactions. We demonstrate that basal-like carcinoma initiation in this model is accompanied by the accumulation of distinct cancer-associated fibroblasts and macrophages and dramatic extracellular matrix remodeling, phenocopying the stromal diversity observed in human triple-negative breast tumors. Dysregulated epithelial-stromal signals were observed, including those mediated by TGF-β, PDGF, and CSF. Autonomous activation of the transcriptional effector TAZ was observed in LATS1/2-deleted cells along with non-autonomous activation within the evolving tumor niche. We further show that inhibition of the YAP/TAZ-associated TEAD family of transcription factors blocks the development of the carcinomas and associated microenvironment. These observations demonstrate that carcinomas resulting from Hippo pathway dysregulation in the mammary epithelium are sufficient to drive cellular events that promote a basal-like tumor-associated niche and suggest that targeting dysregulated YAP/TAZ-TEAD activity may offer a therapeutic opportunity for basal-like mammary tumors.

Genome-wide association studies (GWASs) have identified thousands of variants associated with complex phenotypes, including neuropsychiatric disorders. To better understand their pathogenesis, it is necessary to identify the functional roles of these variants, which are largely located in non-coding DNA regions. Here, we employ a human mesencephalic neuronal cell differentiation model, LUHMES, with sensitive and high-resolution methods to discover enhancers (NET-CAGE), perform DNA conformation analysis (Capture Hi-C) to link enhancers to their target genes, and finally validate selected interactions. We expand the number of known enhancers active in differentiating human LUHMES neurons to 47,350, and find overlap with GWAS variants for Parkinson's disease and schizophrenia. Our findings reveal a fine-tuned regulation of human neuronal differentiation, even between adjacent developmental stages; provide a valuable resource for further studies on neuronal development, regulation, and disorders; and emphasize the importance of exploring the vast regulatory potential of non-coding DNA and enhancers.

Decitabine (DAC), a well-recognized DNA hypomethylating agent, has been applied to treat acute myeloid leukemia. However, clinic investigations revealed that DNA methylation reduction does not correlate with a clinical response, and relapse is prevalent. To gain a better understanding of its anti-tumor mechanism, we perform a temporally resolved CRISPR-Cas9 screen to identify factors governing the DAC response. We show that DNA damage generated by DNMT-DNA adducts and 5-aza-dUTP misincorporation through the dCMP deaminase DCTD act as drivers of DAC-induced acute cytotoxicity. The DNA damage that arises during the next S phase is dependent on DNA replication, unveiling a trans-cell cycle effect of DAC on genome stability. By exploring candidates for synthetic lethality, we unexpectedly uncover that KDM1A promotes survival after DAC treatment through interactions with ZMYM3 and CoREST, independent of its demethylase activity or regulation of viral mimicry. These findings emphasize the importance of DNA repair pathways in DAC response and provide potential biomarkers.