RBM47 is a Critical Regulator of Mouse Embryonic Stem Cell Differentiation.

RNA-binding proteins (RBPs) are pivotal for regulating gene expression as they are involved in each step of RNA metabolism. Several RBPs are essential for viable growth and development in mammals. RNA-binding motif 47 (RBM47) is an RRM-containing RBP whose role in mammalian embryonic development is poorly understood yet deemed to be essential since its loss in mouse embryos leads to perinatal lethality. In this study, we attempted to elucidate the significance of RBM47 in cell-fate decisions of mouse embryonic stem cells (mESCs). Downregulation of Rbm47 did not affect mESC maintenance and the cell cycle but perturbed the expression of primitive endoderm (PrE) markers and increased GATA4 + PrE-like cells. However, the PrE misregulation could be reversed by either overexpressing Rbm47 or treating the knockdown mESCs with the inhibitors of FGFR or MEK, suggesting an implication of RBM47 in regulating FGF-ERK signaling. Rbm47 knockdown affected the multi-lineage differentiation potential of mESCs as it regressed teratoma in NSG mice and led to a skewed expression of differentiation markers in serum-induced monolayer differentiation. Further, lineage-specific differentiation revealed that Rbm47 is essential for proper differentiation of mESCs towards neuroectodermal and endodermal fate. Taken together, we assign a hitherto unknown role(s) to RBM47 in a subtle regulation of mESC differentiation.

NSG-70, a new glioblastoma cell line with mixed proneural-mesenchymal features, associates NOTCH1-WNT5A signaling with stem cell maintenance and angiogenesis.

BACKGROUND: Glioblastoma initiation and progression is believed to be driven by Glioma stem cells (GSCs). Activation of NOTCH1 and WNT, and more recently, non-canonical WNT5A signaling, has been demonstrated to regulate self-renewal and differentiation of the GSCs crucially. High expression levels of NOTCH1 and WNT in GBM tumors contribute to the sustenance of GSCs and mediate characteristic phenotypic plasticity, which is reflected by the different subtypes and tremendous intra-tumor heterogeneity. However, the coregulation of NOTCH1 and WNT5A is not well understood. Here, we studied the role of these molecules in regulating the characteristics of different GSC subtypes. METHODS: We established a novel GSC-enriched cell model, referred to as NSG-70, from a patient with recurrent GBM. NSG-70 cells harbor a unique cytogenetic feature, viz. isochromosome 9q. At the same time, its expression profiles indicate that it is a mixed lineage comprising proneural and mesenchymal subtypes. We examined the relevance of NOTCH1 and WNT5A signaling and their coordinated action in GBM using these cells and other patient-derived models representing different GSC subtypes. RESULTS: Our data revealed that the downregulation of NOTCH1 resulted in the suppression of stem cell and mesenchymal markers and significantly reduced the levels of WNT5A. NOTCH1 knockdown also led to a notable reduction in the vasculogenic mimicry of GSCs. Interestingly, knockdown of WNT5A exhibited similar effects and drove quiescent GSC towards proliferation. In a complementary manner, ectopic expression of WNT5A or rhWNT5A treatment rescued the effects of NOTCH1 knockdown. CONCLUSION: The resistance of GSCs towards conventional therapies in part due to subtype interconversion demands therapies targeting specific GSC subtype. Our study suggests the need for a combinatorial approach that could effectively target the NOTCH1-WNT5A signaling axis toward eliminating GSCs.

Effective Visualization and Easy Tracking of Extracellular Vesicles in Glioma Cells.

Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200-1000 nm) or exosomes (30-200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells and modulate several cellular functions. Being of very small sizes, it is very challenging to analyze them using conventional microscopes. Here, we report a new method developed by us for visualizing EVs using simple immune-fluorescence based technique, wherein the isolated EVs can be stained with fluorescently tagged antibodies to proteins present in EVs. The stained EVs can then be analyzed by using either confocal or super-resolution microscopes. Our method detailed here is equally effective in staining proteins that are present inside the EVs as well as those localized to the membranes of vesicles. By employing unique staining strategies, we have minimized the background noise and thereby improved the signal strength in confocal microscope. Using electron microscopy, we have ascertained that the structural integrity of the labeled EVs is intact. More importantly, the labeling of EVs does not affect their functionality and their localization can be tracked after its uptake by recipient cells without resorting to any conventional reporter-based strategies or lipophilic dyes. In conclusion, the method described here is a simple, sensitive and efficient immune-fluorescence based method for visualization of molecules within the EVs.

Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins.

Long noncoding RNAs constitute a major fraction of the eukaryotic transcriptome, and together with proteins, they intricately fine-tune various growth regulatory signals to control cellular homeostasis. Here, we describe the functional characterisation of a novel pair of long intergenic noncoding RNAs (lincRNAs) comprised of complementary, fully overlapping sense and antisense transcripts Genomic Instability Inducing RNA (Ginir) and antisense RNA of Ginir (Giniras), respectively, from mouse cells. This transcript pair is expressed in a spatiotemporal manner during embryonic development. The individual levels of the sense and antisense transcripts are finely balanced during embryonic growth and in adult tissues. Functional studies of the individual transcripts performed using overexpression and knock-down strategies in mouse cells has led to the discovery that Ginir RNA is a regulator of cellular proliferation and can act as an oncogene having a preeminent role in malignant transformation. Mechanistically, we demonstrate that the oncogenic function of Ginir is mediated by its interaction with centrosomal protein 112 (Cep112). Additionally, we establish here a specific interaction between Cep112 with breast cancer type 1 susceptibility protein (Brca1), another centrosome-associated protein. Next, we prove that the mutual interaction between Cep112 with Brca1 is significant for mitotic regulation and maintenance of genomic stability. Furthermore, we demonstrate that the Cep112 protein interaction with Brca1 protein is impaired when an elevated level of Ginir RNA is present in the cells, resulting in severe deregulation and abnormality in mitosis, leading to malignant transformation. Inhibiting the Ginir RNA function in transformed cells attenuates transformation and restores genomic stability. Together, these findings unravel, to our knowledge, a hitherto-unknown mechanism of oncogenesis mediated by a long noncoding RNA and establishes a unique role of Cep112-Brca1 interaction being modulated by Ginir RNA in maintaining mitotic fidelity.