Mitochondrial regulators are increasingly recognized for their influence on immune signaling within the tumor microenvironment (TME). In glioma, where immunosuppression limits therapeutic efficacy, we investigate how targeting the mitochondrial protein MIRO1 alters the TME. We combine single-nucleus RNA sequencing of murine gliomas treated in vivo with an MIRO1-binding compound and bulk RNA sequencing of human glioma resections treated with the same compound ex vivo. Cross-species transcriptomic integration reveals an MIRO1-responsive program in the TME. Among shared targets, we identify PARP11/Parp11 as a consistently up-regulated gene in glioma, which is down-regulated after MIRO1-binding compound treatment in both human and mouse gliomas. Cell-cell communication analysis shows that a specific cluster of macrophages (MAC1), which exhibits robust Parp11 and Pdl1 (encoding PD-L1) expression, sends immunosuppressive signals to CD8+ cytotoxic T cells, and may receive prostaglandin E2 signals from another cluster of macrophages (MAC4). Targeting MIRO1 eliminates this cell circuitry and reduces the tumor cell population. Our study provides a transcriptomic framework for understanding mitochondria-immune crosstalk and nominates MIRO1-PARP11 as a potential effector axis of brain immune dysfunction.

The cotton seed bug, Oxycarenus hyalinipennis Costa (Hemiptera: Lygaeidae), is an invasive cotton pest detected in California, United States, in 2019. This report details the assembly and annotation of a reference genome for O. hyalinipennis, which will assist with the development of control strategies and provide evolutionary insight into this pest and hemipterans in general. The genome was sequenced from an adult male specimen using PacBio HiFi long reads and Element short-read sequencing of a HiC library. The assembly is 727,196,238 bp long with an N50 of 116 Mb. A total of 28,646 genes were identified by a customized annotation pipeline. Comparative genomics results suggest that chromosomal rearrangements may be common within Pentatomomorpha, based on syntenic analyses across all superfamilies. Comparison between available heteropteran genomes was used to identify changes in gene family size, as well as evolutionary differences, between taxa with different feeding strategies. Notably, herbivorous taxa had significantly larger genomes than predatory taxa. These genomic resources will be useful in efforts to understand and control this pest and advance the understanding of hemipteran evolution.

The NEDD8-activating enzyme (NAE), comprising NAE1 and UBA3, is an anticancer target. The relative contribution of each of these subunits toward NAE inhibitor (NAEi) efficacy remains unclear. We demonstrated a profound interdependence of NAE1 and UBA3 expression. UBA3 reduction augmented NAEi sensitivity, whereas its overexpression led to decreased sensitivity. UBA3 deficiency enhanced RKO xenograft sensitivity to SOMCL-19-133 (NAEi), which was reversed by UBA3 restoration. Cells with naturally low UBA3 expression were highly NAEi-sensitive. The criticality of UBA3 in NAEi sensitivity is not completely unexpected given that the ability of NAEi to directly bind to UBA3 is known. TCGA data showed that rectum adenocarcinoma patients with low UBA3 mRNA had poorer prognoses, and 27.16% of tumors expressed low UBA3 mRNA. We propose that low UBA3 expression may serve as a NAEi sensitivity biomarker, particularly given that MLN4924 (NAEi) phase 3 failures may be due to a lack of patient stratification. Therefore, our key findings, on the criticality of UBA3 in NAEi sensitivity, underpin future clinical evaluations.

Multiciliated cells (MCCs) are specialized cells found in the brain, reproductive and respiratory tracts of mammals, and the epidermis of tadpole-stage Xenopus embryos. KMT5B and KMT5C are histone methyltransferases that deposit the dimethyl mark on histone 4 lysine 20 (H4K20). We previously showed that KMT5B/C double knockdown down-regulates H4K20me2 levels in bulk chromatin, as well as transcription of ciliary genes. MCCs of embryos lacking both enzymes, or only KMT5B, have depleted cilia. Here, we separate the function of KMT5B in multiciliogenesis and show that single knockdown of KMT5B, not KMT5C, leads to aberrant transcription and down-regulation of ciliary genes. This phenotype is rescued by catalytically active PHF8, an H4K20me1 demethylase, whereas hormone-inducible multicilin (MCI), master regulator of cilia, has no effect. Notably, the expression of key transcription factors of ciliogenesis is unaffected by KMT5B depletion, which dominates the transcriptional response to ectopic multicilin. Finally, ATAC-seq in animal caps shows KMT5B knockdown results in few differentially accessible peaks and does not compact chromatin at ciliary genes. This suggests KMT5B regulates MCCs via an alternative pathway to the canonical MCI-driven programme.

Mitochondrial respirometry, the measurement of oxygen consumption rate (OCR) by the electron transport chain (ETC), is a cornerstone of mitochondrial biology and the gold standard for measurements of mitochondrial function. However, existing respirometry methodologies are poorly suited for large-scale studies and high-throughput applications, ultimately limiting the applicability of these methods. This limitation necessitates new methodologies, which are more easily scaled as mitochondrial studies become more complex and diverse. In this study, we detail a respirometry approach we have developed for high-throughput applications including optimized plate layouts, volume-based sample normalization, robust control selection, and automated data processing and quality control. Furthermore, we validate these methodologies across a respirometry study running 703 human brain samples, totaling more than 10,000 data points, which underwent our automated data processing and quality control techniques. Our workflow streamlines assay preparation, execution, and analysis to make respirometry scalable, while reducing operator burden and preserving data integrity. With this study, we provide a transferable blueprint for high-throughput respirometry as the mitochondrial biology field and the studies within it continue to expand in scale.

The majority of pri-miRNAs acquire a 5' cap and 3' poly(A) tail. Mature miRNAs recruit deadenylases that shorten poly(Α) tails triggering target mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a deadenylase that also mediates late steps of noncoding RNA maturation. Herein, we show that PARN affects the expression of a subset of miRNAs in NCI-H520 cells of lung cancer origin, including miR-29a and miR-1207, which are also predicted to target PARN mRNA. PARN associates with pri-miR-29a and pri-miR-1207 regulating their poly(A) lengths. Conversely, miR-29a-3p and miR-1207-5p bind the 3' UTR of PARN mRNA and regulate its expression. Cleavage and polyadenylation specificity factor 6 (CPSF6) recruits PARN to pri-miRNAs and together they affect primary and mature miR-29a-3p levels. Modulation of PARN, miR-29a-3p, or miR-1207-5p expression affects cell migration. We present a model to describe the dynamic relation between PARN and miR-29a and discuss its biological significance.

Regulation of gene expression is essential for neuronal development and function. A prominent regulatory mechanism involves synthesis of proteins at their activity site. Such local protein synthesis enables neurons to respond rapidly and tightly to stimuli. Key components of the translation machinery, including mRNA and ribosomes, were identified in subcellular regions of neurons. Yet, the role of tRNAs and their charging enzymes, aminoacyl-tRNA synthetases (ARS), in this process remains largely unclear. Here, we demonstrate that glycyl-tRNA synthetase (Gars1) mRNA is abundant in neurites and undergoes local translation, producing GARS1 protein. Notably, Gars1 mRNA colocalizes with mitochondria in a translation-dependent manner, with its coding sequence (CDS) sufficient to direct this association. The localized GARS1 protein is in close proximity to tRNAGly, and disrupting their proximity impairs local protein synthesis in neurites. These findings establish the functional importance of GARS1 and tRNAGly in neuritic translation and highlight mitochondria as hubs for mRNA transport and translation.

Nonstructural protein 3 (nsp3) is crucial for SARS-CoV-2 infection. It is the largest protein of the virus with roughly 2000 residues, and a major drug target. However, because of its size, disordered regions, and transmembrane domains, the atomic structure of the whole protein has not yet been established. Only 10 out of its 16 domains were individually determined in experiments. Here, we demonstrate how structural bioinformatics, AI-based fold prediction, and traditional experiments complement each other and can shed light on the makeup of this important protein, both in SARS-CoV-2 and in related viruses. Our method can be generalized for other multidomain proteins. Our prediction-based approach reveals a previously undescribed folded domain, which we could confirm experimentally. Our research also suggests a potential function of the domain Y1: this domain may be involved in the assembly of nsp3, nsp4, and nsp6 into the hexameric pore, which was discovered by electron tomography and exports RNA into the cytosol. The Y1 hexamer, however, could not be expressed on its own. We revise domain segmentation and nomenclature of nsp3 domains.

Dendritic cells (DCs) orchestrate immune responses. Their development is controlled by transcription factors, but epigenetic mechanisms remain poorly understood. DOT1L emerges as a key epigenetic regulator in immune cells. Mapping DOT1L-mediated histone H3K79 methylation in canonical DC subsets revealed that DOT1L modified common and DC subset-specific genes. Deletion of Dot1l in vivo or in vitro decreased myeloid progenitors and increased cDC2s, whereas cDC1s remained unchanged. In addition, in vitro deletion of Dot1l led to loss of plasmacytoid DCs (pDCs) and of IFNα production upon stimulation. Upon in vivo deletion, a decrease in pDCs was only observed after subsequent in vitro expansion. This difference was likely related to insufficient replication-mediated loss of H3K79 methylation in vivo within the time frame studied. Transcriptomes of Dot1l-KO DC subsets exhibited enrichment of antigen presentation pathways, and MHC class II surface levels were up-regulated in pDCs. Mechanistically, inhibition of DOT1L linked the observed effects to its methyltransferase activity. Together, our data indicate that in DCs DOT1L differentially affects the development of canonical subsets and suppresses antigen presentation pathways.

Allergic asthma is characterized by immune dysregulation, and deficiencies in regulatory T-cell (Treg) function are a hallmark of the disease. However, mechanisms of Treg impairment for their therapeutic correction remain poorly defined. The results showed that patient Tregs exhibited a senescent phenotype, including shortened telomeres, increased SA-β-gal activity, and heightened apoptosis. Functionally, they were compromised, showing reduced suppressive capacity and a pro-inflammatory cytokine shift. KQS-1 treatment robustly reversed these defects, restoring FOXP3- and IL-10-dependent Treg suppressive capacity and the production of anti-inflammatory cytokines. This functional rescue centered on these two core Treg signature genes was dependent on Dectin-1 binding and a downstream Raf-1/ROS signaling axis, which drove a sustained epigenetic program characterized by increased H3K4me3 and H3K27ac at the FOXP3 and IL10 loci, focal hypomethylation, and chromatin remodeling at these specific loci. CRISPR-mediated deletion of Dectin-1 abrogated all beneficial effects of KQS-1. As proof of principle that KQS1 is protective, we demonstrate attenuation of airway hyperresponsiveness, inflammation, and remodeling in dust mite-sensitized mice and in recipient mice upon adoptive transfer of KQS-1-trained human Tregs.