Putative Target Sites Research Articles

MicroRNAs miR-186 and miR-150 Down-regulate Expression of the Pro-apoptotic Purinergic P2X7 Receptor by Activation of Instability Sites at the 3′-Untranslated Region of the Gene That Decrease Steady-state Levels of the Transcript

The P2X7 receptor regulates cell growth through mediation of apoptosis. P2X7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X7 mRNA stability. Overexpression of a reporter containing the full-length human P2X7 3'-untranslated region (3'-UTR) or reporters containing parts of the 3'-UTR-P2X7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3'-UTR-P2X7, which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3'-UTR-P2X7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3'-UTR-P2X7.

Cymbidium Ringspot Virus Harnesses RNA Silencing To Control the Accumulation of Virus Parasite Satellite RNA

Cymbidium ringspot virus (CymRSV) satellite RNA (satRNA) is a parasitic subviral RNA replicon that replicates and accumulates at the cost of its helper virus. This 621-nucleotide (nt) satRNA species has no sequence similarity to the helper virus, except for a 51-nt-long region termed the helper-satellite homology (HSH) region, which is essential for satRNA replication. We show that the accumulation of satRNA strongly depends on temperature and on the presence of the helper virus p19 silencing suppressor protein, suggesting that RNA silencing plays a crucial role in satRNA accumulation. We also demonstrate that another member of the Tombusvirus genus, Carnation Italian ringspot virus (CIRV), supports satRNA accumulation at a higher level than CymRSV. Our results suggest that short interfering RNA (siRNA) derived from CymRSV targets satRNA more efficiently than siRNA from CIRV, possibly because of the higher sequence similarity between the HSH regions of the helper and CIRV satRNAs. RNA silencing sensor RNA carrying the putative satRNA target site in the HSH region was efficiently cleaved when transiently expressed in CymRSV-infected plants but not in CIRV-infected plants. Strikingly, replacing the CymRSV HSH box2 sequence with that of CIRV restores satRNA accumulation both at 24 degrees C and in the absence of the p19 suppressor protein. These findings demonstrate the extraordinary adaptation of this virus to its host in terms of harnessing the antiviral silencing response of the plant to control the virus parasite satRNA.

An integrated map of cattle candidate genes for mastitis: a step forward to new genetic markers

To facilitate the development of new genetic markers for mastitis resistance or susceptibility we used genome-wide comparative approach to review all known mastitis-associated loci. We assembled into a map 233 loci that were identified by six different study approaches (QTLs, association studies, expression experiments, AFLP-, miRNA- and epigenetic- studies). To integrate data from different sources and to identify overlapping regions we presented the results in the form of genetic map. The collected data represent genetic background for mastitis-related traits in cattle. Thirty most promising candidate genes (associated with mastitis in different study approaches or at least in two independent studies, or/and overlapping with QTL regions) were selected from database and in silico searched for genetic variability and putative miRNA target sites in 3’UTR. Thirty-one SNPs in the putative promoter/5' UTR (up to 2 kb upstream) of eight candidate genes were found. Bioinformatic analysis revealed that some promoter SNPs might potentially cause gain/loss of the putative transcription factors. Promoter SNPs were also present in CG dinucleotides and therefore possibly involved in gain/loss of CpG sites. In ten mastitis candidate genes we found 56 SNPs in exons of which 21 were non-synonymous substitutions. Additionally, 23 SNPs in intronic regions and 21 SNPs in 3’UTR were found. MiRNA target analysis revealed 89 putative target sites in 18 candidate genes; however, current SNPs were not identified in the miRNA target binding sites or miRNAs expressed in mammary gland. For SNPs with a putative regulatory role found in candidate genes, functional analyses and association studies are needed to facilitate identification of mastitis resistance or susceptibility alleles possibly involved in mastitis regulatory pathways.

Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3' untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3'UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3'UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3'UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.

Cloning and characterization of an acyl‐CoA‐dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site‐directed mutagenesis to modify enzyme activity and oil content

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Let-7 regulates Dicer expression and constitutes a negative feedback loop

microRNAs (miRNA) are small, endogenously expressed non-coding RNAs that are sequentially processed by Drosha and Dicer from primary transcripts, by negatively regulating the expression of protein-coding genes through either translational repression or RNA degradation. Their expression patterns are developmentally regulated and/or tissue specific, while altered expressions of certain miRNAs are frequently observed in human cancers, though the underlying regulatory mechanism is largely unknown. Herein, we show that Dicer expression was inversely correlated with expression levels of mature let-7 in a panel of human cancer cell lines, showing association with cell growth and cell cycle phases. Overexpression of let-7 significantly reduced the expression of Dicer at both the protein and messenger RNA levels, whereas antisense-mediated reduction of let-7 expression conversely increased Dicer at both levels. A luciferase assay using a reporter carrying a putative target site in the 3' untranslated region of Dicer revealed that let-7 directly affects Dicer expression. Downregulation of Dicer resulted in a reduced expression of mature let-7. Furthermore, overexpression of let-7 decreased the levels of expression of other mature miRNAs, while knockdown of let-7 increased those levels. Taken together, these findings strongly suggest the possible existence of a novel regulatory loop, in which let-7 may play a role as a key miRNA for implementing the tightly regulated, equilibrated state of Dicer and various miRNAs.

Identification of SNPs located in putative microRNA target region of six functional genes in chickens through bioinformatic analysis

GDF-8, IGF-I, IGF-III, IGF2R, IGFBP2, and GHR are candidate genes affecting important economic trait in chicken. The putative microRNA target sites of 3' untranslated region of these genes were identified by means of specialized algorithms (miRanda and TargetScan), and the candidate SNPs located in the microRNA target region were also identified. Approximately 125 candidate SNPs were found throughout the 26 putative microRNA target regions in six gene 3'-UTR. Among the 125 SNPs, 47 were located in the microRNA targets, 44 and 35 were located in 5'and 3'flanking regions which equalled to the size of the given target. Twelve of the 47 candidates were located in the match of the microRNA seed. These SNPs, which were located in the match of the microRNA seed and 3 ' flanking regions may affect microRNA regulation and contribute to poultry phenotypic variation.

Threonine 66 in the death domain of IRAK-1 is critical for interaction with signaling molecules but is not a target site for autophosphorylation

Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.

RNAplex: a fast tool for RNA–RNA interaction search

Regulatory RNAs often unfold their action via RNA-RNA interaction. Transcriptional gene silencing by means of siRNAs and miRNA as well as snoRNA directed RNA editing rely on this mechanism. Additionally ncRNA regulation in bacteria is mainly based upon RNA duplex formation. Finding putative target sites for newly discovered ncRNAs is a lengthy task as tools for cofolding RNA molecules like RNAcofold and RNAup are too slow for genome-wide search. Tools like RNAhybrid that neglects intramolecular interactions have runtimes proportional to O(m x n), albeit with a large prefactor. Still in many cases the need for even faster methods exists. We present a new program, RNAplex, especially designed to quickly find possible hybridization sites for a query RNA in large RNA databases. RNAplex uses a slightly different energy model which reduces the computational time by a factor 10-27 compared to RNAhybrid. In addition a length penalty allows to focus the target search on short highly stable interactions. RNAplex can be downloaded at http://www.tbi.univie.ac.at/~htafer/ Supplementary data are available at Bioinformatics online.

MiR-148 targets human DNMT3b protein coding region

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer) myostatin gene.

BackgroundMyostatin (MSTN) is a member of the transforming growth factor-β superfamily that negatively regulates growth of skeletal muscle tissue. The gene encoding for the MSTN peptide is a consolidate candidate for the enhancement of productivity in terrestrial livestock. This gene potentially represents an important target for growth improvement of cultured finfish.ResultsHere we report molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer) MSTN-1 gene. The barramundi MSTN-1 was encoded by three exons 379, 371 and 381 bp in length and translated into a 376-amino acid peptide. Intron 1 and 2 were 412 and 819 bp in length and presented typical GT...AG splicing sites. The upstream region contained cis-regulatory elements such as TATA-box and E-boxes. A first assessment of sequence variability suggested that higher mutation rates are found in the 5' flanking region with several SNP's present in this species. A putative micro RNA target site has also been observed in the 3'UTR (untranslated region) and is highly conserved across teleost fish. The deduced amino acid sequence was conserved across vertebrates and exhibited characteristic conserved putative functional residues including a cleavage motif of proteolysis (RXXR), nine cysteines and two glycosilation sites. A qualitative analysis of the barramundi MSTN-1 expression pattern revealed that, in adult fish, transcripts are differentially expressed in various tissues other than skeletal muscles including gill, heart, kidney, intestine, liver, spleen, eye, gonad and brain.ConclusionOur findings provide valuable insights such as sequence variation and genomic information which will aid the further investigation of the barramundi MSTN-1 gene in association with growth. The finding for the first time in finfish MSTN of a miRNA target site in the 3'UTR provides an opportunity for the identification of regulatory mutations on the expression of this gene.

MicroRNA-373 induces expression of genes with complementary promoter sequences

Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.

A Universal Framework for Regulatory Element Discovery across All Genomes and Data Types

Deciphering the noncoding regulatory genome has proved a formidable challenge. Despite the wealth of available gene expression data, there currently exists no broadly applicable method for characterizing the regulatory elements that shape the rich underlying dynamics. We present a general framework for detecting such regulatory DNA and RNA motifs that relies on directly assessing the mutual information between sequence and gene expression measurements. Our approach makes minimal assumptions about the background sequence model and the mechanisms by which elements affect gene expression. This provides a versatile motif discovery framework, across all data types and genomes, with exceptional sensitivity and near-zero false-positive rates. Applications from yeast to human uncover putative and established transcription-factor binding and miRNA target sites, revealing rich diversity in their spatial configurations, pervasive co-occurrences of DNA and RNA motifs, context-dependent selection for motif avoidance, and the strong impact of posttranscriptional processes on eukaryotic transcriptomes.

The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes

The microRNAs miR-1 and miR-133 are preferentially expressed in cardiac and skeletal muscles and have been shown to regulate differentiation and proliferation of these cells. We report here a novel aspect of cellular function of miR-1 and miR-133 regulation of cardiomyocyte apoptosis. miR-1 and miR-133 produced opposing effects on apoptosis, induced by oxidative stress in H9c2 rat ventricular cells, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. miR-1 level was significantly increased in response to oxidative stress. We identified single target sites for miR-1 only, in the 3'-untranslated regions of the HSP60 and HSP70 genes, and multiple putative target sites for miR-133 throughout the sequence of the caspase-9 gene. miR-1 reduced the levels of HSP60 and HSP70 proteins without changing their transcript levels, whereas miR-133 did not affect HSP60 and HSP70 expression at all. By contrast, miR-133 repressed caspase-9 expression at both the protein and mRNA levels. The post-transcriptional repression of HSP60 and HSP70 and caspase-9 was further confirmed by luciferase reporter experiments. Our results indicate that miR-1 and miR-133 are involved in regulating cell fate with increased miR-1 and/or decreased miR-133 levels favoring apoptosis and decreased miR-1 and/or miR-133 levels favoring survival. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of caspase-9 by miR-133 contributes significantly to their opposing actions.

Mir-29 regulates Mcl-1 protein expression and apoptosis

Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis.

MicroRNA miR-133 Represses HERG K+ Channel Expression Contributing to QT Prolongation in Diabetic Hearts

We have previously found that the ether-a-go-go related gene (ERG), a long QT syndrome gene encoding a key K(+) channel (I(Kr)) in cardiac cells, is severely depressed in its expression at the protein level but not at the mRNA level in diabetic subjects. The mechanisms underlying the disparate alterations of ERG protein and mRNA, however, remained unknown. We report here a remarkable overexpression of miR-133 in hearts from a rabbit model of diabetes, and in parallel the expression of serum response factor (SRF), which is known to be a transactivator of miR-133, was also robustly increased. Delivery of exogenous miR-133 into the rabbit myocytes and cell lines produced post-transcriptional repression of ERG, down-regulating ERG protein level without altering its transcript level and caused substantial depression of I(Kr), an effect abrogated by the miR-133 antisense inhibitor. Functional inhibition or gene silencing of SRF down-regulated miR-133 expression and increased I(Kr) density. Repression of ERG by miR-133 likely underlies the differential changes of ERG protein and transcript thereby depression of I(Kr), and contributes to repolarization slowing thereby QT prolongation and the associated arrhythmias, in diabetic hearts. Our study provided the first evidence for the pathological role of miR-133 in adult hearts and thus expanded our understanding of the cellular function and pathophysiological roles of miRNAs.

Protein Kinase Cα-dependent Phosphorylation of the mRNA-stabilizing Factor HuR: Implications for Posttranscriptional Regulation of Cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

Inference of miRNA targets using evolutionary conservation and pathway analysis.

BackgroundMicroRNAs have emerged as important regulatory genes in a variety of cellular processes and, in recent years, hundreds of such genes have been discovered in animals. In contrast, functional annotations are available only for a very small fraction of these miRNAs, and even in these cases only partially.ResultsWe developed a general Bayesian method for the inference of miRNA target sites, in which, for each miRNA, we explicitly model the evolution of orthologous target sites in a set of related species. Using this method we predict target sites for all known miRNAs in flies, worms, fish, and mammals. By comparing our predictions in fly with a reference set of experimentally tested miRNA-mRNA interactions we show that our general method performs at least as well as the most accurate methods available to date, including ones specifically tailored for target prediction in fly. An important novel feature of our model is that it explicitly infers the phylogenetic distribution of functional target sites, independently for each miRNA. This allows us to infer species-specific and clade-specific miRNA targeting. We also show that, in long human 3' UTRs, miRNA target sites occur preferentially near the start and near the end of the 3' UTR.To characterize miRNA function beyond the predicted lists of targets we further present a method to infer significant associations between the sets of targets predicted for individual miRNAs and specific biochemical pathways, in particular those of the KEGG pathway database. We show that this approach retrieves several known functional miRNA-mRNA associations, and predicts novel functions for known miRNAs in cell growth and in development.ConclusionWe have presented a Bayesian target prediction algorithm without any tunable parameters, that can be applied to sequences from any clade of species. The algorithm automatically infers the phylogenetic distribution of functional sites for each miRNA, and assigns a posterior probability to each putative target site. The results presented here indicate that our general method achieves very good performance in predicting miRNA target sites, providing at the same time insights into the evolution of target sites for individual miRNAs. Moreover, by combining our predictions with pathway analysis, we propose functions of specific miRNAs in nervous system development, inter-cellular communication and cell growth. The complete target site predictions as well as the miRNA/pathway associations are accessible on the ElMMo web server.

PolymiRTS Database: linking polymorphisms in microRNA target sites with complex traits

Polymorphism in microRNA Target Site (PolymiRTS) database is a collection of naturally occurring DNA variations in putative microRNA target sites. PolymiRTSs may affect gene expression and cause variations in complex phenotypes. The database integrates sequence polymorphism, phenotype and expression microarray data, and characterizes PolymiRTSs as potential candidates responsible for the quantitative trait locus (QTL) effects. It is a resource for studying PolymiRTSs and their implications in phenotypic variations. PolymiRTS database can be accessed at .

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.