Noncanonical function of DGCR8 controls mESC exit from pluripotency

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. The analysis of newly generated mutant mESCs revealed that DGCR8 is essential for the exit from the pluripotency state. To dissociate canonical versus noncanonical functions of DGCR8, we complemented the mutant mESCs with a phosphomutant DGCR8, which restored microRNA levels but did not rescue the exit from pluripotency defect. Integration of omics data and RNA immunoprecipitation experiments established DGCR8 as a direct interactor of Tcf7l1 mRNA, a core component of the pluripotency network. Finally, we found that DGCR8 facilitated the splicing of Tcf7l1, an event necessary for the differentiation of mESCs. Our data reveal a new noncanonical function of DGCR8 in the modulation of the alternative splicing of Tcf7l1 mRNA in addition to its established function in microRNA biogenesis

miR-33 regulates cell proliferation, cell cycle progression and liver regeneration

Der Cholesterin-Stoffwechsel ist sehr streng auf zellulärer Ebene reguliert und ist essentiell für das Zellwachstum. MicroRNAs (miRNAs), eine Klasse nicht-kodierender RNAs, wurden als kritische Regulatoren der Genexpression identifiziert und entfalten ihre Wirkung vorwiegend auf posttranskriptioneller Ebene. Aktuelle Arbeiten aus der Gruppe um Fernández-Hernando haben gezeigt, dass hsa-miR-33a und hsa-miR-33b, miRNAs die in den Intronsequenzen der Gene für die Sterol-regulatorischen Element- Bindungsproteine (SREBP-2 und SREBP -1) lokalisiert sind, den Cholesterin-Stoffwechsel im Einklang mit ihren Wirtsgenen regulieren. Gleichermaßen inhibiert miR-33 Schlüsselenzyme in der Regulation der Fettsäureoxidation, einschließlich CROT, CPT1A, HADHB, SIRT6, AMPKα, genauso wie IRS2, eine wesentliche Komponente des Insulin-Signalwegs in der Leber. Diese Studie zeigt, dass hsa-miR-33 Familienmitglieder nicht nur Gene in Cholesterin- und Fettsäure-Stoffwechsel sowie Insulin-Signalwege regulieren, sondern zusätzlich die Expression von Genen des Zellzyklus und der Zellproliferation modulieren. miR-33 inhibiert die Expression der CDK6 und CCND1, wodurch sowohol die Zellproliferation als auch die Zellzyklusprogression verringert wird. Die Überexpression von miR-33 induziert einen signifikanten G1 Zellzyklusarrest. Durch eine Inhibierung der miR-33 Expression mittels 2''F/MOE-modifiziert Phosphorothioat-Backbone Antisense-Oligonukleotiden, wird die Leberregeneration nach partieller Hepatektomie (PH) in Mäusen verbessert, was auf eine wichtige Rolle für miR-33 in der Regulation der Hepatozytenproliferation während der Leberregeneration hinweist. Zusammengefasst zeigen diese Daten, dass Srebf/miR-33 Locus kooperieren, um Zellproliferation und Zellzyklusprogression zu regulieren, und könnte somit auch relevant für die menschliche Leberregeneration sein.Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. Cellular imbalances of cholesterol and fatty acid metabolism lead to pathological processes, including atherosclerosis and metabolic syndrome. MicroRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression acting predominantly at posttranscriptional level. Recent work from Fernández-Hernando´s group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the sterol regulatory element-binding protein (SREBP-2 and SREBP-1) genes, respectively, regulate cholesterol metabolism in concert with their host genes. Similarly, miR-33 targets key enzymes involved in the regulation of fatty acid oxidation including CROT, CPT1A, HADHB, SIRT6 and AMPKα, likewise, IRS2, an essential component of the insulin- signaling pathway in the liver. This study shows that hsa-miR-33 family members not only regulate genes involved in cholesterol and fatty acid metabolism and insulin signaling, but in addition modulate the expression of genes involved in cell cycle regulation and cell proliferation. Thus, miR-33 inhibited the expression of CDK6 and CCND1, thereby reducing cell proliferation and cell cycle progression. Over-expression of miR-33 induced a significant G1 cell cycle arrest and most importantly, inhibition of miR-33 expression using 2’F/MOE-modified phosphorothioate backbone antisense oligonucleotides improved liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these data establish that Srebf/miR-33 locus may co-operate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration

Membrane-tethered mucins and lubricin mediate the interfacial properties of model ocular epithelial surfaces

The human ocular surface is enriched with endogenously expressed mucins, which are known to contribute to its lubricious character. Reduced biosynthesis or loss of functional mucins has been reported in some dry eye disease (DED) patients, contributing to mechanical alterations during blink cycles that can result in tissue damage and vision-threatening sequelae. While identifying strategies to reduce adhesion and shear stresses at the ocular surface is a promising approach to improve the signs and symptoms of DED, current pre-clinical models generally rely on scarce, heterogeneous tissue samples or model synthetic substrates that do not capture the complex biochemical and biophysical cues present at the ocular surface. Here we developed a mucin-deficient dry eye mimetic cell model and utilized contact angle hysteresis and step-strain rheological tests to investigate the contributions of mucins and mucin-like glycoproteins to the interfacial, rheological, and adhesive properties of ocular epithelial surfaces. This model system was designed with the purpose of providing mechanistic insight into the consequences of ocular surface mucin dysfunction that may inform treatment strategies. The contact angle hysteresis measurements showed that the hydration of the model ocular epithelial surfaces is maintained even in absence of endogenous membrane-tethered mucins. However, stress relaxation behaviors at the interface of model ocular surfaces demonstrated that membrane-tethered mucins are essential for biolubrication at the model interface. Supplementation with recombinant human lubricin, a mucin-like glycoprotein currently undergoing clinical trials for DED treatment, restored the lubrication function induced by the lack of cell surface mucins in a dose-dependent manner. This suggests that lubrication-related dysfunction due to mucin deficiency may be reversible and that recombinant human lubricin might be a promising treatment for DED patients. Together, these results demonstrate that mucin-deficient, biomimetic ocular surfaces serve as a promising platform for drug screening assays and fundamental studies of ocular surface biology

The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes

Complex gene regulation systems ensure the maintenance of cellular identity during early development in mammals. Eukaryotic small RNAs have emerged as critical players in RNA interference (RNAi) by mediating gene silencing during embryonic stem cell self-renewal. Most of the proteins involved in the biogenesis of small RNAs are essential for proliferation and differentiation into the three germ layers of mouse embryonic stem cells. In the last decade, new functions for some RNAi proteins, independent of their roles in RNAi pathways, have been demonstrated in different biological systems. In parallel, new concepts in stem cell biology have emerged. Here, we review and integrate the current understanding of how RNAi proteins regulate stem cell identity with the new advances in the stem cell field and the recent non-canonical functions of the RNAi proteins. Finally, we propose a reevaluation of all RNAi mutant phenotypes, as non-canonical (small non-coding RNA independent) functions may contribute to the molecular mechanisms governing mouse embryonic stem cells commitment.ISSN:0022-2836ISSN:1089-863

A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Anti-Adhesion Properties, and Barrier Functionality

Dry eye disease (DED) affects more than 100 million people worldwide, causing significant patient discomfort and imposing a multi-billion-dollar burden on global health care systems. In DED patients, the natural biolubrication process that facilitates pain-free blinking goes awry due to an imbalance of lipid, aqueous medium, and mucin in the tear film, resulting in ocular surface damage. Identifying strategies to reduce adhesion and shear stresses between the ocular surface and the conjunctival cells lining the inside of the eyelid during blink cycles is a promising approach to improve the signs and symptoms of DED. However, current preclinical models for screening ocular lubricants rely on scarce, heterogeneous tissue samples or model substrates that do not capture the complex biochemical and biophysical cues present at the ocular surface. To recapitulate the hierarchical architecture and phenotype of the ocular interface for preclinical drug screening, we developed an in vitro mucin-deficient DED model platform that mimics the complexity of the ocular interface and investigated its utility in biolubrication, anti adhesion, and barrier protection studies using recombinant human lubricin, a promising investigational therapy for DED. The biomimetic platform recapitulated the pathological changes in biolubrication, adhesion, and barrier functionality often observed in mucin-deficient DED patients and demonstrated that recombinant human lubricin can reverse the damage induced by mucin loss in a dose- and conformation-dependent manner. Taken together, these results highlight the potential of the platform—and recombinant human lubricin—in advancing the standard of care for mucin deficient DED patients

Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells

In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.ISSN:2213-671

Control of Cholesterol Metabolism and Plasma High-Density Lipoprotein Levels by microRNA-144

RATIONALE: Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including ABCA1 and ABCG1. ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. OBJECTIVE: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. METHODS AND RESULTS: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed a miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to ApoA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 appears to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. CONCLUSIONS: 1) miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression; and 2) modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease