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Long noncoding RNAs (lncRNAs) play essential roles in the development and progression of many cancers. However, the contributions of lncRNAs to medulloblastoma (MB) remain poorly understood. Here, we identify Miat as an lncRNA enriched in the sonic hedgehog group of MB that is required for maintenance of a treatment-resistant stem-like phenotype in the disease. Loss of Miat results in the differentiation of tumor-initiating, stem-like MB cells and enforces the differentiation of tumorigenic stem-like MB cells into a nontumorigenic state. Miat expression in stem-like MB cells also facilitates treatment resistance by down-regulating p53 signaling and impairing radiation-induced cell death, which can be reversed by therapeutic inhibition of Miat using antisense oligonucleotides. Mechanistically, the RNA binding protein Metadherin (Mtdh), previously linked to resistance to cytotoxic therapy in cancer, binds to Miat in stem-like MB cells. Like the loss of Miat, the loss of Mtdh reduces tumorigenicity and increases sensitivity to radiation-induced death in stem-like MB cells. Moreover, Miat and Mtdh function to regulate the biogenesis of several microRNAs and facilitate tumorigenesis and treatment resistance. Taken together, these data reveal an essential role for the lncRNA Miat in sustaining a treatment-resistant pool of tumorigenic stem-like MB cells.

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

BackgroundCRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis.ResultsWe present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription.ConclusionsTaken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

The article discusses the importance of engaging in conversations with Holocaust survivors, rather than simply collecting their stories as sources of knowledge. It highlights the significance of building relationships and friendships with survivors, emphasizing the value of learning together through sustained conversations. The text also explores various perspectives on survivor testimonies, challenging traditional approaches to analyzing and interpreting their accounts. The authors stress the human connection and emotional depth involved in understanding survivors' experiences, advocating for a more personal and empathetic approach to Holocaust studies. [Extracted from the article]

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that [GJA1.sup.M213L/M213L] mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous [GJA1.sup.M213L/WT] mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart., Introduction Intercellular communication between cardiomyocytes via connexin-43 (Cx43) gap junctions enables the rapid spread of electrical signals and synchronous ventricular contraction. Oligomerization of 6 Cx43 molecules gives rise to a [...]

Group 2 innate lymphoid cells (ILC2s) produce high levels of IL-5 and IL-13, both of which are important pathogenic mediators in eosinophilic esophagitis (EoE). ILC2s have not been previously described in EoE. Our study demonstrates the novel finding that ILC2s are increased in esophageal biopsies from EoE patients with active disease compared with inactive EoE and non-diseased controls, implicating these cells in EoE pathogenesis.