microRNA Recognition Elements Research Articles

Inhibition of pro-inflammatory signaling in human primary macrophages by enhancing arginase-2 via target site blockers

The modulation of macrophage phenotype from a pro-inflammatory to an anti-inflammatory state holds therapeutic potential in the treatment of inflammatory disease. We have previously shown that arginase-2 (Arg2), a mitochondrial enzyme, is a key regulator of the macrophage anti-inflammatory response. Here, we investigate the therapeutic potential of Arg2 enhancement via target site blockers (TSBs) in human macrophages. TSBs are locked nucleic acid antisense oligonucleotides that were specifically designed to protect specific microRNA recognition elements (MREs) in human ARG2 3' UTR mRNA. TSBs targeting miR-155 (TSB-155) and miR-3202 (TSB-3202) MREs increased ARG2 expression in human monocyte-derived macrophages. This resulted in decreased gene expression and cytokine production of TNF-α and CCL2 and, for TSB-3202, in an increase in the anti-inflammatory macrophage marker, CD206. Proteomic analysis demonstrated that a network of pro-inflammatory responsive proteins was modulated by TSBs. In silico bioinformatic analysis predicted that TSB-3202 suppressed upstream pro-inflammatory regulators including STAT-1 while enhancing anti-inflammatory associated proteins. Proteomic data were validated by confirming increased levels of sequestosome-1 and decreased levels of phosphorylated STAT-1 and STAT-1 upon TSB treatment. In conclusion, upregulation of Arg2 by TSBs inhibits pro-inflammatory signaling and is a promising novel therapeutic strategy to modulate inflammatory signaling in human macrophages.

Development of a Beta Cell-Specific Expression Control Element for Recombinant Adeno-Associated Virus.

Diabetes mellitus, caused by loss or dysfunction of the insulin-producing beta cells of the pancreas, is a promising target for recombinant adeno-associated virus (rAAV)-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell type-specific expression control element is needed. In this study, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INS × 2) and microRNA (miRNA) recognition elements (MREs). The INS × 2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2 × -MRE expression vector was highly specific to human beta cells and stem cell-derived beta cells.

Prediction of single-nucleotide polymorphisms within microRNAs binding sites of neuronal genes related to multiple sclerosis: A preliminary study

Background:Different genetic variants, including the single-nucleotide polymorphisms (SNPs) present in microRNA recognition elements (MREs) within 3'UTR of genes, can affect miRNA-mediated gene regulation and susceptibility to a variety of human diseases such as multiple sclerosis (MS), a disease of the central nervous system. Since the expression of many genes associated with MS is controlled by microRNAs (miRNAs), the aim of this study was to analyze SNPs within miRNA binding sites of some neuronal genes associated with MS.Materials and Methods:Fifty-seven neuronal genes related to MS were achieved using dbGaP, DAVID, DisGeNET, and Oviddatabases. 3'UTR of candidate genes were assessed for SNPs, and miRNAs' target prediction databases were used for predicting miRNA binding sites. Results:Three hundred and eight SNPs (minor allele frequency >0.05) were identified in miRNA binding sites of 3'UTR of 44 genes. Among them, 42 SNPs in 22 genes had miRNA binding sites and miRNA prediction tools suggested 71 putative miRNAs binding sites on these genes. Moreover, in silico analysis predicted 22 MRE-modulating SNPs and 22 MRE-creating SNPs in the 3'UTR of these candidate genes.Conclusions:These candidate MRE-SNPs can alter miRNAs binding sites and mRNA gene regulation. Therefore, these genetic variants and miRNAs might be involved in MS susceptibility and pathogenesis and hence would be valuable for further functional verification investigation.

SUN-718 Reciprocal Regulation of miR-375 and ICER in Pancreatic Beta Cells

MicroRNA-375 (miR-375) is overexpressed in people with type 2 diabetes (T2D) and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both are regulated by the cAMP pathway. This overexpression of miR-375 in T2D led us to hypothesize that beta cells with elevated and reduced levels of miR-375 will result in decreased and increased glucose-stimulated insulin secretion (GSIS), respectively. Results showed that when miR-375 was overexpressed, GSIS decreased by 61% when compared to a control in 25 mM glucose. Results showed that when miR-375 was inhibited, GSIS increased 6% when compared to a control in 25 mM glucose. In human islets, we found that inhibiting miR-375 led to an average 19% increase in GSIS, though due to the variability of human tissue these data were not significant (N=5). To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. HEK-293 (human embryonic kidney) cells that were transfected with a luciferase reporter plasmid containing a cAMP recognition element (CRE) and a plasmid driving the overexpression of ICER had a 75% decrease when compared to our control (P<0.05). When transiently-expressed ICER was knocked down via siRNA, promoter activity increased by 13.1-fold (P< 0.05). Using a chromatin immunoprecipitation assay we found that an ICER antibody pulled down the rat miR-375 promoter an average of 13-fold compared with a control antibody (N=2). Additionally, because of a sequence alignment showing possible binding of miR-375 to the human ICER transcript we hypothesize that the two are in a negative feedback loop and can regulate each other’s expression. To investigate the double negative feedback loop a plasmid was constructed containing the GFP reporter gene and either the human or rodent ICER 3’UTR predicted miR-375 binding site. The GFP reporter assay was conducted to determine if miR-375 binds to ICER’s microRNA recognition element (MRE) in a species-specific way. In our GFP reporter experiment, data shows there is a 50% reporter gene decrease between our negative control and the human ICER MRE (N=4, P=0.017). Understanding microRNA gene regulation in the pancreas may have important implications for patients with T2D. MiR-375 may have the ability to interact with human ICER in a double negative feedback loop in the cAMP second messenger pathway, which will further clarify cellular mechanisms to potentially improve T2D drugs.

MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis.

Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute-2(AGO2)-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. These microRNAs increased in cells plated on stiff vs. soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements (MREs) within the 3’UTRs of CAM mRNAs. Inhibition of DROSHA or AGO2, or disruption of MREs within individual target mRNAs such as Connective Tissue Growth Factor (CTGF), induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix (ECM) deposition in the zebrafish fin-fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.

A novel class of microRNA-recognition elements that function only within open reading frames.

MicroRNA (miRNA) are well known to target 3’ untranslated regions (3’UTR) in mRNAs to silence gene expression at post-transcriptional levels. Multiple reports have also indicated the capability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function in a similar mechanism(s) regardless of the location of their action sites. We herein report a class of miRNA recognition elements (MREs) that exclusively function in CDS regions in humans. Through functional and mechanistic characterization of these “unusual” MREs, we demonstrate that CDS-targeted miRNAs require extensive base pairings in the 3’ side rather than the 5’ seed; cause gene silencing in an Argonaute-dependent, but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences for miRNAs to target CDS versus 3’UTR and suggest that CDS-targeted miRNAs may enlist a translational quality control (QC)-related mechanism to regulate translation in mammalian cells.

Pseudogenes Provide Evolutionary Evidence for the Competitive Endogenous RNA Hypothesis.

The competitive endogenous RNA (ceRNA) hypothesis is an attractively simple model to explain the biological role of many putatively functionless noncoding RNAs. Under this model, there exist transcripts in the cell whose role is to titrate out microRNAs such that the expression level of another target sequence is altered. That it is logistically possible for expression of one microRNA recognition element (MRE)-containing transcript to affect another is seen in the multiple examples of pathogenic effects of inappropriate expression of MRE-containing RNAs. However, the role, if any, of ceRNAs in normal biological processes and at physiological levels is disputed. By comparison of parent genes and pseudogenes we show, both for a specific example and genome-wide, that the pseudo-3′ untranslated regions (3′UTRs) of expressed pseudogenes are frequently retained and are under selective constraint in mammalian genomes. We found that the pseudo-3′UTR of BRAFP1, a previously described oncogenic ceRNA, has reduced substitutions relative to its pseudo-coding sequence, and we show sequence constraint on MREs shared between the parent gene, BRAF, and the pseudogene. Investigation of RNA-seq data reveals expression of BRAFP1 in normal somatic tissues in human and in other primates, consistent with biological ceRNA functionality of this pseudogene in nonpathogenic cellular contexts. Furthermore, we find that on a genome-wide scale pseudo-3′UTRs of mammalian pseudogenes (n = 1,629) are under stronger selective constraint than their pseudo-coding sequence counterparts, and are more often retained and expressed. Our results suggest that many human pseudogenes, often considered nonfunctional, may have an evolutionarily constrained role, consistent with the ceRNA hypothesis.

Transcriptomic analysis of high-throughput sequencing about circRNA, lncRNA and mRNA in bladder cancer

An increasing number of studies have revealed that long noncoding RNA (lncRNA) and circular RNA (circRNA) participate in the carcinogenesis and progression of tumors. However, most of these noncoding RNAs are of unknown function or without annotation. We carried out high-throughput sequencing to investigate the differential expression of lncRNAs and circRNAs and their biological functions in four coupled bladder cancer and adjacent noncancerous tissues. We identified significant differentially expressed transcripts and genes and acquired their annotations from the RefSeq and circBase databases, then confirmed the expression of randomly selected RNAs with quantitative real-time PCR. We also constructed a coding-noncoding co-expression (CNC) network and a competing endogenous RNA (ceRNA) network to predict the functions of these RNAs using well-studied protein-coding mRNA. Compared with adjacent tissues, 56 lncRNAs, 34 circRNAs and 467 protein-coding mRNAs were upregulated while 32 lncRNAs, 84 circRNAs and 326 protein-coding mRNAs were downregulated in cancer tissues. Co-expression analysis showed that expression of LINC00885 were correlated with GATA3 expression. The ceRNA network indicated that lncRNA MIR194-2HG, AATBC and circRNA PGM5 could harbor bladder cancer-related microRNA (miRNA) recognition elements. We performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to ascertain the biological function of significantly dysregulated genes. Cell cycle and cell division pathways related to proliferation and apoptosis were obvious in enriched terms. Comprehensive analysis indicated that the dysregulated lncRNAs and circRNAs could participate in the genesis and progression of bladder cancer. Our approach may therefore be valuable for detecting novel transcripts, discovering new biomarkers for bladder cancer and expounding the pathogenic mechanisms of this disease.

RNA helicase Mov10 is essential for gastrulation and central nervous system development.

Mov10 is an RNA helicase that modulates access of Argonaute 2 to microRNA recognition elements in mRNAs. We examined the role of Mov10 in Xenopus laevis development and show a critical role for Mov10 in gastrulation and in the development of the central nervous system (CNS). Knockdown of maternal Mov10 in Xenopus embryos using a translation blocking morpholino led to defects in gastrulation and the development of notochord and paraxial mesoderm, and a failure to neurulate. RNA sequencing of the Mov10 knockdown embryos showed significant upregulation of many mRNAs when compared with controls at stage 10.5 (including those related to the cytoskeleton, adhesion, and extracellular matrix, which are involved in those morphogenetic processes). Additionally, the degradation of the miR-427 target mRNA, cyclin A1, was delayed in the Mov10 knockdowns. These defects suggest that Mov10's role in miRNA-mediated regulation of the maternal to zygotic transition could lead to pleiotropic effects that cause the gastrulation defects. Additionally, the knockdown of zygotic Mov10 showed that it was necessary for normal head, eye, and brain development in Xenopus consistent with a recent study in the mouse. Mov10 is essential for gastrulation and normal CNS development. Developmental Dynamics 247:660-671, 2018. © 2017 Wiley Periodicals, Inc.

Neuronal-expressed microRNA-targeted pseudogenes compete with coding genes in the human brain

MicroRNAs orchestrate brain functioning via interaction with microRNA recognition elements (MRE) on target transcripts. However, the global impact of potential competition on the microRNA pool between coding and non-coding brain transcripts that share MREs with them remains unexplored. Here we report that non-coding pseudogene transcripts carrying MREs (PSG+MRE) often show duplicated origin, evolutionary conservation and higher expression in human temporal lobe neurons than comparable duplicated MRE-deficient pseudogenes (PSG−MRE). PSG+MRE participate in neuronal RNA-induced silencing complexes (RISC), indicating functional involvement. Furthermore, downregulation cell culture experiments validated bidirectional co-regulation of PSG+MRE with MRE-sharing coding transcripts, frequently not their mother genes, and with targeted microRNAs; also, PSG+MRE single-nucleotide polymorphisms associated with schizophrenia, bipolar disorder and autism, suggesting interaction with mental diseases. Our findings indicate functional roles of duplicated PSG+MRE in brain development and cognition, supporting physiological impact of the reciprocal co-regulation of PSG+MRE with MRE-sharing coding transcripts in human brain neurons.

Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine.

Small noncoding microRNAs act as post-transcriptional regulators of gene expression involved in diverse biologic functions. Pregnane X receptor (PXR, NR1I2), a member of the superfamily of nuclear receptors, is a transcription factor governing the transport and biotransformation of various drugs and other chemicals. In the present study, we identified a specific microRNA (miR) involved in regulating the expression and functionality of human PXR (hPXR). According to bioinformatics analysis employing three commonly used algorithms (TargetScan, miRanda, and DIANA-microT-CDS), miR-18a-5p was predicted to be the top candidate microRNA regulator of hPXR. Consequently, this microRNA was selected for detailed experimental investigation. As shown in cell-based dual-luciferase reporter gene assays, functional interaction occurred between miR-18a-5p and the microRNA recognition element of miR-18a-5p in the 3'-untranslated region of hPXR mRNA. Transfection of LS180 human colorectal adenocarcinoma cells with an miR-18a-5p mimic decreased hPXR mRNA and protein expression, whereas transfection of LS180 cells with an miR-18a-5p inhibitor increased hPXR mRNA and protein expression. The decrease in hPXR expression by the miR-18a-5p mimic was associated with a reduction in the extent of hPXR target gene (CYP3A4) induction by rifampin and rilpivirine. Treatment of untransfected LS180 cells with either of these hPXR agonists decreased endogenous expression of miR-18a-5p, and this preceded the onset of CYP3A4 induction. In conclusion, miR-18a-5p is a negative regulator of hPXR expression and the hPXR agonists rifampin and rilpivirine are chemical suppressors of miR-18a-5p expression.

Identification and Analysis of P53-Mediated Competing Endogenous RNA Network in Human Hepatocellular Carcinoma.

Recent studies have indicated that long non-coding RNAs (lncRNAs) and mRNA function as competing endogenous RNAs (ceRNAs) that compete to bind to shared microRNA (miRNA) recognition elements (MREs) to perform specific biological functions during tumorigenesis. The tumor suppressor p53 is a master regulator of cancer-related biological processes by acting as a transcription factor to regulate target genes including miRNA and lncRNA. However, the mechanism in human hepatocellular carcinoma and whether p53-mediated RNA targets could form ceRNA network remain unclear. Here, we identified a series of differential expressed miRNAs, lncRNA and mRNA which were potentially regulated by p53 using RNA sequencing in HepG2. Genomic characteristics comparative analysis showed significant differences between mRNAs and lncRNAs. By integrating experimentally confirmed Ago2 and p53 binding sites, we constructed a highly reliable p53-mediated ceRNA network using hypergeometric test. The KEGG pathway enrichment analysis showed that the ceRNA network highly enriched in the cancer or p53-associated signaling pathways. Finally, using betweenness centrality analysis, we identified five master miRNAs (hsa-miR-3620-5p, hsa-miR-3613-3p, hsa-miR-6881-3p, hsa-miR-6087 and hsa-miR-18a-3p) that regulated most of the target RNAs, suggesting these miRNAs play central roles in the whole p53-mediated ceRNAs network. Taken together, our results provide a new regulatory mechanism of p53 networks for future studies in cancer therapeutics.

S.17.03 - Long non-coding pseudogene transcripts compete with mRNAs that share microRNA recognition elements with them in human brain neurons

S.17.03 - Long non-coding pseudogene transcripts compete with mRNAs that share microRNA recognition elements with them in human brain neurons

The long intergenic non-coding RNA CCR492 functions as a let-7 competitive endogenous RNA to regulate c-Myc expression

In mammals the cell-cycle progression through the G1 phase is a tightly regulated process mediated by the transcriptional activation of early genes in response to mitogenic stimuli, whose dysregulation often leads to tumorigenesis. We here report the discovery by RNA-seq of cell-cycle regulated (CCR) long intergenic non-coding RNAs (lincRNAs), potentially involved in the control of the cell-cycle progression. We identified 10 novel lincRNAs expressed in response to serum treatment in mouse embryonic fibroblasts (MEFs) and in BALB/c fibroblasts, comparably to early genes. By loss-of-function experiments we found that lincRNA CCR492 is required for G1/S progression, localizes in the cell cytoplasm and contains 4 let-7 microRNA recognition elements (MREs). Mechanistically, CCR492 functions as a competing endogenous RNA (ceRNA) to antagonize the function of let-7 microRNAs, leading to the de-repression of c-Myc. Moreover, we show that ectopic expression of CCR492 along with a constitutively active H-Ras promotes cell transformation. Thus, we identified a new lincRNA expressed as an early gene in mammalian cells to regulate the cell-cycle progression by upregulating c-Myc expression.

CYP2A7 pseudogene transcript affects CYP2A6 expression in human liver by acting as a decoy for miR-126.

Human cytochrome P450 (CYP)2A6 is responsible for the metabolic activation of tobacco-related nitrosamines, as well as the metabolism of nicotine and some pharmaceutical drugs. There are large interindividual differences in CYP2A6 activity and expression, largely attributed to genetic polymorphisms. However, the variability was observed within homozygotes of the wild-type CYP2A6 gene. In this study, we investigated the possibility that CYP2A6 might be regulated by microRNA. A luciferase assay revealed that a microRNA recognition element (MRE) of miR-126* found in the 3'-untranslated region (UTR) of CYP2A6 mRNA is functional. We established two HEK293 cell lines stably expressing CYP2A6, with one including and the other excluding the full-length 3'-UTR (HEK/2A6+UTR and HEK/2A6 cells, respectively). Overexpression of miR-126* markedly decreased CYP2A6 protein levels, enzyme activity, and mRNA level in HEK/2A6+UTR cells, whereas it marginally decreased those in HEK/2A6 cells, indicating that the 3'-UTR including the MRE is functional for the downregulation of CYP2A6 by miR-126*. The inhibition of miR-126* increased CYP2A6 protein levels in primary human hepatocytes, suggesting that miR-126* downregulates endogenous CYP2A6 expression. In 20 human liver samples, the expression ratios of CYP2A6 and a pseudogene transcript CYP2A7 mRNA were highly variable (CYP2A7/CYP2A6: 0.1 to 12). Interestingly, we found that CYP2A7 was another target of miR-126* and restored the miR-126*-dependent downregulation of CYP2A6 by acting as a decoy for miR-126*. In conclusion, this study demonstrates that human CYP2A6 is post-transcriptionally regulated by miR-126* and that CYP2A7 affects CYP2A6 expression by competing for miR-126* binding.

Altered viral replication and cell responses by inserting microRNA recognition element into PB1 in pandemic influenza A virus (H1N1) 2009.

Objective. MicroRNAs (miRNAs) are endogenous noncoding RNAs that spatiotemporally modulate mRNAs in a posttranscriptional manner. Engineering mutant viruses by inserting cell-specific miRNA recognition element (MRE) into viral genome may alter viral infectivity and host responses in vital tissues and organs infected with pandemic influenza A virus (H1N1) 2009 (H1N1pdm). Methods. In this study, we employed reverse genetics approach to generate a recombinant H1N1pdm with a cell-specific miRNA target sequence inserted into its PB1 genomic segment to investigate whether miRNAs are able to suppress H1N1pdm replication. We inserted an MRE of microRNA-let-7b (miR-let-7b) into the open reading frame of PB1 to test the feasibility of creating a cell-restricted H1N1pdm virus since let-7b is abundant in human bronchial epithelial cells. Results. miR-let-7b is rich in human bronchial epithelial cells (HBE). Incorporation of the miR-let-7b-MRE confers upon the recombinant H1N1pdm virus susceptibility to miR-let-7b targeting, suggesting that the H1N1pdm and influenza A viruses can be engineered to exert the desired replication restrictive effect and decrease infectivity in vital tissues and organs. Conclusions. This approach provides an additional layer of biosafety and thus has great potential for the application in the rational development of safer and more effective influenza viral vaccines.

MOV10 and FMRP Regulate AGO2 Association with MicroRNA Recognition Elements

The fragile X mental retardation protein FMRP regulates translation of its bound mRNAs through incompletely defined mechanisms. FMRP has been linked to the microRNA pathway, and we show here that it associates with the RNA helicase MOV10, also associated with the microRNA pathway. FMRP associates with MOV10 directly and in an RNA-dependent manner and facilitates MOV10's association with RNAs in brain and cells, suggesting a cooperative interaction. We identified the RNAs recognized by MOV10 using RNA immunoprecipitation and iCLIP. Examination of the fate of MOV10 on RNAs revealed a dual function for MOV10 in regulating translation: it facilitates microRNA-mediated translation of some RNAs, but it also increases expression of other RNAs by preventing AGO2 function. The latter subset was also bound by FMRP in close proximity to the MOV10 binding site, suggesting that FMRP prevents MOV10-mediated microRNA suppression. We have identified a mechanism for FMRP-mediated translational regulation through its association with MOV10.

Genome-wide association study combined with biological context can reveal more disease-related SNPs altering microRNA target seed sites

BackgroundEmerging studies demonstrate that single nucleotide polymorphisms (SNPs) resided in the microRNA recognition element seed sites (MRESSs) in 3′UTR of mRNAs are putative biomarkers for human diseases and cancers. However, exhaustively experimental validation for the causality of MRESS SNPs is impractical. Therefore bioinformatics have been introduced to predict causal MRESS SNPs. Genome-wide association study (GWAS) provides a way to detect susceptibility of millions of SNPs simultaneously by taking linkage disequilibrium (LD) into account, but the multiple-testing corrections implemented to suppress false positive rate always sacrificed the sensitivity. In our study, we proposed a method to identify candidate causal MRESS SNPs from 12 GWAS datasets without performing multiple-testing corrections. Alternatively, we used biological context to ensure credibility of the selected SNPs.ResultsIn 11 out of the 12 GWAS datasets, MRESS SNPs were over-represented in SNPs with p-value ≤ 0.05 (odds ratio (OR) ranged from 1.1 to 2.4). Moreover, host genes of susceptible MRESS SNPs in each of the 11 GWAS dataset shared biological context with reported causal genes. There were 286 MRESS SNPs identified by our method, while only 13 SNPs were identified by multiple-testing corrections with a given threshold of 1 × 10−5, which is a common cutoff used in GWAS. 27 out of the 286 candidate SNPs have been reported to be deleterious while only 2 out of 13 multiple-testing corrected SNPs were documented in PubMed. MicroRNA-mRNA interactions affected by the 286 candidate SNPs were likely to present negatively correlated expression. These SNPs introduced greater alternation of binding free energy than other MRESS SNPs, especially when grouping by haplotypes (4210 vs. 4105 cal/mol by mean, 9781 vs. 8521 cal/mol by mean, respectively).ConclusionsMRESS SNPs are promising disease biomarkers in multiple GWAS datasets. The method of integrating GWAS p-value and biological context is stable and effective for selecting candidate causal MRESS SNPs, it reduces the loss of sensitivity compared to multiple-testing corrections. The 286 candidate causal MRESS SNPs provide researchers a credible source to initialize their design of experimental validations in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-669) contains supplementary material, which is available to authorized users.

Post-Transcriptional Regulation of Interferons and Their Signaling Pathways

Interferons (IFNs) are low molecular weight cell-derived proteins that include the type I, II, and III IFN families. IFNs are critical for an optimal immune response during microbial infections while dysregulated expression can lead to autoimmune diseases. Given its role in disease, it is important to understand cellular mechanisms of IFN regulation. 3' untranslated regions (3' UTRs) have emerged as potent regulators of mRNA and protein dosage and are controlled through multiple regulatory elements including adenylate uridylate (AU)-rich elements (AREs) and microRNA (miRNA) recognition elements. These AREs are targeted by RNA-binding proteins (ARE-BPs) for degradation and/or stabilization through an ARE-mediated decay process. miRNA are endogenous, single-stranded RNA molecules ~22 nucleotides in length that regulate mRNA translation through the miRNA-induced silencing complex. IFN transcripts, like other labile mRNAs, harbor AREs in their 3' UTRs that dictate the turnover of mRNA. This review is a survey of the literature related to IFN regulation by miRNA, ARE-BPs, and how these complexes interact dynamically on the 3' UTR. Additionally, downstream effects of these post-transcriptional regulators on the immune response will be discussed. Review topics include past studies, current understanding, and future challenges in the study of post-transcriptional regulation affecting IFN responses.

Human-specific microRNA regulation of FOXO1: implications for microRNA recognition element evolution

MicroRNAs (miRNAs) have been established as important negative post-transcriptional regulators for gene expression. Within the past decade, miRNAs targeting transcription factors (TFs) has emerged as an important mechanism for gene expression regulation. Here, we tested the hypothesis that in TF 3'UTRs, human-specific single nucleotide change(s) that create novel miRNA recognition elements (MREs) contribute to species-specific differences in TF expression. From several potential human-specific TF MREs, one candidate, a member of the Forkhead Box O (FOXO) subclass in the Forkhead family known as Forkhead Box O1 (FOXO1; FKHR; NM_002015) was tested further. Human FOXO1 contains two sites predicted to confer miR-183-mediated post-transcriptional regulation: one specific to humans and the other conserved. Utilizing dual luciferase expression reporters, we show that only the human FOXO1 3'UTR contains a functional miR-183 site, not found in chimpanzee or mouse 3'untranslated regions (UTRs). Site-directed mutagenesis supports functionality of the human-specific miR-183 site, but not the conserved miR-183 site. Via overexpression and target site protection assays, we show that human FOXO1 is regulated by miR-183, but mouse FOXO1 is not. Finally, FOXO1-regulated cellular phenotypes, including cell invasion and proliferation, are impacted by miR-183 targeting only in human cells. These results provide strong evidence for human-specific gain of TF MREs, a process that may underlie evolutionary differences between phylogenic groups.