N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, ...and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic β-cells exposed to inflammatory stimuli.
mRNA stability influences gene expression and translation in almost all living organisms, and the levels of mRNA molecules in the cell are determined by a balance between production and decay. ...Maintaining an accurate balance is crucial for the correct function of a wide variety of biological processes and to maintain an appropriate cellular homeostasis. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of gene expression through different molecular mechanisms, including mRNA stabilization. In this review we provide an overview on the molecular mechanisms by which lncRNAs modulate mRNA stability and decay. We focus on how lncRNAs interact with RNA binding proteins and microRNAs to avoid mRNA degradation, and also on how lncRNAs modulate epitranscriptomic marks that directly impact on mRNA stability.
Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that ...long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6‐methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele‐specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803‐YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.
The increasing interest on RNA based therapeutics make long noncoding RNAs (lncRNAs) appealing targets for the development of novel treatments. Herein we describe the allele‐specific and N6‐methyladenosine (m6A) methylation dependent ability of LOC339803 lncRNA to regulate the expression of proinflammatory cytokines in the intestine. Deregulated LOC339803 levels in patients with intestinal inflammation and results from ex vivo LOC339803 reduction experiments point to this lncRNA as an interesting therapeutic target for intestinal inflammatory disorders.
Type 1 diabetes (T1D) is a complex autoimmune disease that develops in genetically susceptible individuals. Most T1D‐associated single nucleotide polymorphisms (SNPs) are located in non‐coding ...regions of the human genome. Interestingly, SNPs in long non‐coding RNAs (lncRNAs) may result in the disruption of their secondary structure, affecting their function, and in turn, the expression of potentially pathogenic pathways. In the present work, the function of a virus‐induced T1D‐associated lncRNA named ARGI (Antiviral Response Gene Inducer) is characterized. Upon a viral insult, ARGI is upregulated in the nuclei of pancreatic β cells and binds to CTCF to interact with the promoter and enhancer regions of IFNβ and interferon‐stimulated genes, promoting their transcriptional activation in an allele‐specific manner. The presence of the T1D risk allele in ARGI induces a change in its secondary structure. Interestingly, the T1D risk genotype induces hyperactivation of type I IFN response in pancreatic β cells, an expression signature that is present in the pancreas of T1D patients. These data shed light on the molecular mechanisms by which T1D‐related SNPs in lncRNAs influence pathogenesis at the pancreatic β cell level and opens the door for the development of therapeutic strategies based on lncRNA modulation to delay or avoid pancreatic β cell inflammation in T1D.
Long non‐coding RNAs (lncRNAs) are emerging as regulators for several molecular processes that are important for human diseases. Herein, the function of a type 1 diabetes (T1D)‐associated lncRNA named ARGI is described. ARGI modulates the transcription of virus‐induced IFNβ and interfer on stimulated genes (ISGs) in pancreatic β cells in an allele‐specific manner.
Local heat generation from magnetic nanoparticles (MNPs) exposed to alternating magnetic fields can revolutionize cancer treatment. However, the application of MNPs as anticancer agents is limited by ...serious drawbacks. Foremost among these are the fast uptake and biodegradation of MNPs by cells and the unpredictable magnetic behavior of the MNPs when they accumulate within or around cells and tissues. In fact, several studies have reported that the heating power of MNPs is severely reduced in the cellular environment, probably due to a combination of increased viscosity and strong NP agglomeration. Herein, we present an optimized protocol to coat magnetite (Fe3O4) NPs larger than 20 nm (FM-NPs) with high molecular weight PEG molecules that avoid collective coatings, prevent the formation of large clusters of NPs and keep constant their high heating performance in environments with very different ionic strengths and viscosities (distilled water, physiological solutions, agar and cell culture media). The great reproducibility and reliability of the heating capacity of this FM-NP@PEG system in such different environments has been confirmed by AC magnetometry and by more conventional calorimetric measurements. The explanation of this behavior has been shown to lie in preserving as much as possible the magnetic single domain-type behavior of nearly isolated NPs. In vitro endocytosis experiments in a colon cancer-derived cell line indicate that FM-NP@PEG formulations with PEGs of higher molecular weight (20 kDa) are more resistant to endocytosis than formulations with smaller PEGs (5 kDa), showing quite large uptake mean-life (τ > 5 h) in comparison with other NP systems. The in vitro magnetic hyperthermia was performed at 21 mT and 650 kHz during 1 h in a pre-endocytosis stage and complete cell death was achieved 48 h posthyperthermia. These optimal FM-NP@PEG formulations with high resistance to endocytosis and predictable magnetic response will aid the progress and accuracy of the emerging era of theranostics.
Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that ...long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N
-methyladenosine (m
A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m
A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.
Abstract
Long noncoding RNAs have been identified in most vertebrates, but the functional characterization of these molecules is challenging, mainly due to the lack of linear sequence homology ...between species. In this work, we aimed to find functional evolutionary convergent lncRNAs involved in development by screening of k-mer content (nonlinear similarity) and secondary structure-based approaches combining in silico, in vitro and in vivo validation analysis. From the Madagascar gecko genes, we have found a non-orthologous lncRNA with a similar k-mer content and structurally concordant with the human lncRNA
EVX1AS
. Analysis of function-related characteristics together with locus-specific targeting of human
EVX1AS
and gecko
EVX1AS-like
(i.e., CRISPR Display) in human neuroepithelial cells and chicken mesencephalon have confirmed that gecko
EVX1AS-like
lncRNA mimics human
EVX1AS
function and induces
EVX1
expression independently of the target species. Our data shows functional convergence of non-homologous lncRNAs and presents a useful approach for the definition and manipulation of lncRNA function within different model organisms.
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that develops due to the interaction between genetic and environmental factors. More than 160 loci ...have been associated with IBD, but the functional implication of many of the associated genes remains unclear. N6-Methyladenosine (m6A) is the most abundant internal modification in mRNA. m6A methylation regulates many aspects of mRNA metabolism, playing important roles in the development of several pathologies. Interestingly, SNPs located near or within m6A motifs have been proposed as possible contributors to disease pathogenesis. We hypothesized that certain IBD-associated SNPs could regulate the function of genes involved in IBD development via m6A-dependent mechanisms. We used online available GWAS, m6A and transcriptome data to find differentially expressed genes that harbored m6A-SNPs associated with IBD. Our analysis resulted in five candidate genes corresponding to two of the major IBD subtypes:
and
for Crohn's Disease and
,
and
for Ulcerative Colitis. Further analysis using in silico predictions and co-expression analyses in combination with in vitro functional studies showed that our candidate genes seem to be regulated by m6A-dependent mechanisms. These findings provide the first indication of the implication of RNA methylation events in IBD pathogenesis.
Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function ...of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified.
Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis.
We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release.
These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.
Medulloblastoma is the most common and malignant pediatric brain tumor in childhood. It originates from dysregulation of cerebellar development, due to an excessive proliferation of cerebellar ...granule neuron precursor cells (CGNPs). The underlying molecular mechanisms, except for the role of SHH and WNT pathways, remain largely unknown. ERBB4 is a tyrosine kinase receptor whose activity in cancer is tissue dependent. In this study, we characterized the role of ERBB4 during cerebellum development and medulloblastoma progression paying particular interests to its role in CGNPs and medulloblastoma stem cells (MBSCs). Our results show that ERBB4 is expressed in the CGNPs during cerebellum development where it plays a critical role in migration, apoptosis and differentiation. Similarly, it is enriched in the population of MBSCs, where also controls those critical processes, as well as self-renewal and tumor initiation for medulloblastoma progression. These results are translated to clinical samples where high levels of ERBB4 correlate with poor outcome in Group 4 and all medulloblastomas groups. Transcriptomic analysis identified critical processes and pathways altered in cells with knock-down of ERBB4. These results highlight the impact and underlying mechanisms of ERBB4 in critical processes during cerebellum development and medulloblastoma.
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