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Background: Long non-coding RNAs (lncRNAs) can perform tasks of key relevance in fat cells, contributing, when defective, to the burden of obesity and its sequelae. Here, scrutiny of adipose tissue transcriptomes before and after bariatric surgery (GSE53378) granted identification of 496 lncRNAs linked to the obese phenotype. Only expression of linc-GALNTL6-4 displayed an average recovery over 2-fold and FDR-adjusted p-value

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.

Abstract 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.

IntroductionMost 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.MethodsHere, 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.ResultsWe 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.ConclusionsThese 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.

Abstract The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24–2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

Immune-mediated diseases, such as celiac disease, type 1 diabetes or multiple sclerosis, are a clinically heterogeneous group of diseases that share many key genetic triggers. Although the pathogenic mechanisms responsible for the development of immune mediated disorders is not totally understood, high-throughput genomic studies, such as GWAS and Immunochip, performed in the past few years have provided intriguing hints about underlying mechanisms and pathways that lead to disease. More than a hundred gene variants associated with disease susceptibility have been identified through such studies, but the progress toward understanding the underlying mechanisms has been slow. The majority of the identified risk variants are located in non-coding regions of the genome making it difficult to assign a molecular function to the SNPs. However, recent studies have revealed that many of the non-coding regions bearing disease-associated SNPs generate long non-coding RNAs (lncRNAs). LncRNAs have been implicated in several inflammatory diseases, and many of them have been shown to function as regulators of gene expression. Many of the disease associated SNPs located in lncRNAs modify their secondary structure, or influence expression levels, thereby affecting their regulatory function, hence contributing to the development of disease.

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.

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: UBE2L3 and SLC22A4 for Crohn’s Disease and TCF19, C6orf47 and SNAPC4 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.

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.

The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.