Putative Target Sites Research Articles

Abstract 18008: Post-Transcriptional Regulation of Adiponectin by Micro-RNA 378 in Human Visceral and Subcutaneous Adipose Tissue

Purpose: MicroRNAs (miR), single-stranded non-protein coding gene products, regulate gene expression through post-transcriptional inhibition, and involved in essential biological processes including obesity and insulin resistance. miR-378/378*, which are encoded by PGC-1β gene and counterbalance the metabolic actions of PGC-1β. We conducted to elucidate the role of miR378 expression in human adiposity and adipose tissue inflammation. Methods: Study 1 (clinical study): Pair samples were obtained from subcutaneous (SAT) and visceral adipose tissue (VAT) during elective operation in 71 men and 42 women. MiRNA and adipocytokines levels were determined by qRT-PCR. Study 2 (in vitro study): We quantitated adipocytokine transcription during adipogenesis in 3T3-L1 cells overexpressing mimics or inhibitors of miR378 and assessed post-transcriptional regulation in HEK 293 cells expressing a renilla-luciferase-adiponectin-3’UTR sequence. Results: Study 1: The mean size, median and mode of adipocytes were negatively correlated with levels of adipose adiponectin in SAT and in VAT. CD68 was negatively, and TLR4, PPARγ2, PGC-1β, and ESRRG were positively correlated with adipose adiponectin. Study 2: Upon in silico search, we found a putative target site for miRNA378/378* in the 3 prime untranslated region (3’ UTR) of adiponectin gene. Expression levels of miR378 as well as adiponectin, ACSL1, GPAT6, PPARγ2 and PGC1β were increased in 3T3L1 during differentiation. The expression of adiponectin and ESRRG, a PGC-1β partner, were inhibited by miR378 mimics. Luminescence activity in HEK 293 cells expressing a renilla-luciferase-adiponectin-3’UTR sequence was inhibited by overexpressing mimics of miR378 and the decrease was recovered by adding the inhibitors of miR378, indicating that the posttranscriptional activity of adiponectin 3’UTR of was regulated via the miR378 binding site. Conclusion: There were depot- and size-dependent variations in adiponectin, miRNA378 and its related genes in SAT and VAT. Roles of miR378 and other possible miRNAs were warranted in adiponectin expression and its post-translational modification, which links to metabolic and cardiovascular abnormalities associated with obesity and insulin resistance.

MicroRNA-138 Regulates Hypoxia-Induced Endothelial Cell Dysfunction By Targeting S100A1

The Ca2+ sensor S100A1 is essential for proper endothelial cell (EC) nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown. Here we show that ECs transfected with a S100A1–3′ untranslated region (UTR) luciferase reporter construct display significantly reduced gene expression when subjected to low oxygen levels or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (MiR-138) could target the 3′UTR of S100A1. Patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased MiR-138 levels and decreased S100A1 protein expression. Consistent with this finding, hypoxia greatly increased MiR-138 levels in ECs, but not in skeletal muscle C2C12 myoblasts or differentiated myotubes or primary human vascular smooth muscle cells. Transfection of a MiR-138 mimic into ECs reduced S100A1–3 ‘UTR reporter gene expression, while transfection of an anti MiR-138 prevented the hypoxia-induced downregulation of the reporter gene. Deletion of the 22 nucleotide putative MiR-138 target site abolished the hypoxia-induced loss of reporter gene expression. Knockdown of Hif1-α mediated by siRNA prevented loss of hypoxia-induced reporter gene expression. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression even in the absence of hypoxia. Finally, primary ECs transfected with a MiR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. We conclude that hypoxia-induced MiR-138 is an essential mediator of EC dysfunction via its ability to target the 3′UTR of S100A1.

Modeling the binding specificity of the RNA-binding protein GLD-1 suggests a function of coding region–located sites in translational repression

To understand the function of the hundreds of RNA-binding proteins (RBPs) that are encoded in animal genomes it is important to identify their target RNAs. Although it is generally accepted that the binding specificity of an RBP is well described in terms of the nucleotide sequence of its binding sites, other factors such as the structural accessibility of binding sites or their clustering, to enable binding of RBP multimers, are also believed to play a role. Here we focus on GLD-1, a translational regulator of Caenorhabditis elegans, whose binding specificity and targets have been studied with a variety of methods such as CLIP (cross-linking and immunoprecipitation), RIP-Chip (microarray measurement of RNAs associated with an immunoprecipitated protein), profiling of polysome-associated mRNAs and biophysical determination of binding affinities of GLD-1 for short nucleotide sequences. We show that a simple biophysical model explains the binding of GLD-1 to mRNA targets to a large extent, and that taking into account the accessibility of putative target sites significantly improves the prediction of GLD-1 binding, particularly due to a more accurate prediction of binding in transcript coding regions. Relating GLD-1 binding to translational repression and stabilization of its target transcripts we find that binding sites along the entire transcripts contribute to functional responses, and that CDS-located sites contribute most to translational repression. Finally, biophysical measurements of GLD-1 affinity for a small number of oligonucleotides appear to allow an accurate reconstruction of the sequence specificity of the protein. This approach can be applied to uncover the specificity and function of other RBPs.

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

MiR-15a is underexpressed and inhibits the cell cycle by targeting CCNE1 in breast cancer

MicroRNAs (miRNAs) are a small class of non-coding RNAs that are widely dysregulated in various cancers. They act as either oncogenes or tumor suppressor genes in human cancer. The purpose of this study was to examine the expression levels of miR-15a in human breast cancer and its potential role in disease pathogenesis. The expression levels of miR-15a were measured by quantitative polymerase chain reaction (qPCR) in 40 breast cancer specimens and adjacent normal breast tissues. MTT assays, colony formation assays, transwell chamber migration assays, cell cycle and apoptosis assays were used to explore the potential function of miR-15a in MDA-MB-231 human breast cancer cells. Luciferase reporter assays were performed to validate the regulation of a putative target of miR-15a, in corroboration with qPCR and western blot assays. We found that the expression of miR-15a was lower in breast cancer specimens compared with adjacent normal tissues (p<0.05). Overexpression of miR-15a inhibited cellular growth, suppressed migration and arrested cells at the G1 phase, but did not promote cellular apoptosis. Luciferase assays indicated that miR-15a can bind with its putative target site in the 3'-untranslated region (3'-UTR) of CCNE1, suggesting that CCNE1 is a direct target of miR-15a. qPCR and western blot analysis indicated that the overexpression of miR-15a results in the downregulation of CCNE1 at the mRNA and protein levels. Taken together, the upregulation of miR-15a expression causes cellular growth inhibition, suppression of migration and G1 phase arrest by targeting CCNE1. These findings suggest that miR-15a may act as a tumor suppressor gene in breast cancer and that, in the future, it could be used as a therapeutic target for the treatment of breast cancer.

Structural and functional characterisation of the αS1-casein (CSN1S1) gene and association studies with milk traits in Assaf sheep breed

Genetic polymorphisms in genes encoding milk proteins are of interest to the animal breeding and dairy industry due to their effects on production traits, milk composition and milk quality. For this purpose, the total length of the CSN1S1 gene has been sequenced and characterised in Assaf animals with an extreme phenotype for milk protein content. In the present study, some of the polymorphisms were genotyped for association analysis studies with milk protein content in a population of 444 Assaf ewes. Furthermore, the influences of these polymorphisms on the transcription rate of this gene were also evaluated in 45 Churra Tensina ewes. An 18427-bp DNA-sequence of the CSN1S1 gene, including the entire CDS, 5′- and 3′UTR regions, and the promoter region, was analysed, leading to the identification of 61 polymorphisms. Two polymorphisms detected in the 5′ flanking region were located within possible trans-acting factor binding sites, modifying the putative CdxA and GATA-1 consensus sites. The SNP (JN560175: g.1123C>A) that modifies a putative CdxA consensus site showed a significant effect on the expression of the CSN1S1 gene (P=0.043), but no effect was found for protein content. A SNP in exon 17 (g.16101C>T) produces a non-conservative substitution of isoleucine to threonine at position 209 in the pre-protein (GenBank accession no. JN560175), but no significant associations with milk protein content were found. Finally, two SNPs located in exons 18 (g.16721C>A) and 19 (g.17826G>A) modify a putative target site for two bovine miRNA (bta-miR-631 and bta-miR-1224, respectively). Only the SNP in exon 18 showed a lightly significant additive genetic effect (P<0.1), with protein content for the CC genotype (4.43%) that accounts for 2.37% of the population mean (4.33%) for this trait. The results of the association and expression studies in exon 18 (g.16721C>A) were consistent, as the expression of the CSN1S1 gene in animals with the CC genotype was approximately 1.8 times greater than the expression of the GG genotype. Further studies concerning cheese yield and casein content as well as functional studies of transcription factors and miRNA binding activity are needed to elucidate the function of these SNPs and their application to breeding schemes.

Nuclear Receptors and microRNA-144 Coordinately Regulate Cholesterol Efflux

The ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cellular cholesterol efflux and HDL maturation. ABCA1 mRNA has an unusually long 3’ untranslated region, which makes it highly susceptible to microRNA (miRNA) targeting and repression. As such, multiple miRNAs have been reported to directly target ABCA1, including miR-33a/b, miR-26, miR-106b, and miR-758. Many of these miRNAs participate in feed-forward or feedback networks in controlling cholesterol and lipid homeostasis. Antisense oligonucleotide-based inhibition of miR-33 was found to increase HDL-C levels and regress atherosclerosis in mice and non-human primates. In this edition of Circulation Research, two separate studies identified novel miRNA networks driven by nuclear receptor induced miR-144 targeting of ABCA1 and cholesterol efflux. The first study reports that miR-144 serves to buffer uncontrolled ABCA1 activation in response to high cholesterol states and liver X receptor (LXR) activation in macrophages and liver. The second study highlights the role of miR-144 and ABCA1 in promotion of bile acid secretion in response to farensoid X receptor (FXR) activation in the liver. These studies suggest that anti-miR-144, like anti-miR-33, could be a novel approach to targeting HDL and reverse cholesterol transport.

Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families

The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5'RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.

MiR‐139 Inhibits Mcl‐1 Expression and Potentiates TMZ‐Induced Apoptosis in Glioma

Mcl-1, an antiapoptotic member of the Bcl-2 family, is overexpressed in human glioblastoma, conferring a survival advantage to tumor cells. The mechanisms underlying its dysregulation have not been clarified. In this study, we explored the involvement of micro-RNAs that acted as endogenous sequence-specific suppressors of gene expression. Using computational and TCGA analysis, we identified miR-139 as being downregulated in glioblastoma in comparison with human brain tissue, as well as possessing a putative target site in Mcl-1 mRNA. Overexpression of miR-139 led to a clear decrease in Mcl-1 expression in gliomas. Reporter assays revealed direct post-transcriptional regulation involving miR-139 and the 3'-untranslated region of Mcl-1. Human glioma tissues with low expression of miR-139 displayed higher expression of Mcl-1 protein than those with high expression, suggesting that low miR-139 contributes to Mcl-1 overexpression. In addition, upregulation of miR-139 suppressed the proliferation and enhanced temozolomide (TMZ)-induced apoptosis. Finally, we observed that Mcl-1 knockdown resulted in similar effects compared with miR-139 transfection. Our results suggested that miR-139 negatively regulated Mcl-1 and induced apoptosis in cooperation with an anticancer drug TMZ in glioma.

MicroRNA-9 regulates the expression of peroxisome proliferator-activated receptor δ in human monocytes during the inflammatory response

PPARδ is involved in the inflammatory response and its expression is induced by cytokines, however, limited knowledge has been produced regarding its regulation. Since recent findings have shown that microRNAs, which are small non-coding RNAs that regulate gene expression, are involved in the immune response, we set out to investigate whether PPARδ can be regulated by microRNAs expressed in monocytes. Bioinformatic analysis identified a putative miR-9 target site within the 3′-UTR of PPARδ that was subsequently verified to be functional using reporter constructs. Primary human monocytes stimulated with LPS showed a downregulation of PPARδ and its target genes after 4 h while the expression of miR-9 was induced. Analysis of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages showed that human PPARδ mRNA as well as miR-9 expression was higher in M1 compared to M2 macrophages. Furthermore, treatment with the PPARδ agonist, GW501516, induced the expression of PPARδ target genes in the pro-inflammatory M1 macrophages while no change was observed in the anti-inflammatory M2 macrophages. Taken together, these data suggest that PPARδ is regulated by miR-9 in monocytes and that activation of PPARδ may be of importance in M1 pro-inflammatory but not in M2 anti-inflammatory macrophages in humans.

MiRNA-26b inhibits proliferation by targeting PTGS2 in breast cancer

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs (20–24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. The aim of this study was to investigate the expression pattern of microRNA-26b (miR-26b) in human breast cancer, and its potential role in disease pathogenesis.MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-26b in 38 breast cancer specimens and adjacent normal breast tissues. MTT assays were conducted to explore the impact of miR-26b overexpression on the proliferation of human MDA-MB-231 breast cancer cells. Luciferase reporter assays were employed to validate regulation of a putative target of miR-26b. The effect of modulating miR-26b on endogenous levels of this target were subsequently confirmed via qRT-PCR and Western blot.ResultsMiR-26b expression was relatively decreased in breast cancer specimens compared with adjacent normal tissues (P<0.01). Overexpression of miR-26b suppressed MDA-MB-231 cell growth. Luciferase assays using a reporter carrying a putative miR-26b target site in the 3' untranslated region of PTGS2 revealed that miR-26b directly targets PTGS2. Overexpression of miR-26b led to downregulation of PTGS2 at the mRNA and protein level, as assessed by qRT-PCR and Western blot. Targeted knockdown of PTGS2 by siRNA significantly inhibited the proliferation of MDA-MB-231 breast cancer cells.ConclusionsMiR-26b may act as a tumor suppressor in breast cancer. The overexpression of miR-26b inhibits cellular growth by targeting PTGS2, suggesting its use as a potential therapeutic target for breast cancer.

A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing

BackgroundMicroRNAs (miRNAs) play a critical role in down-regulating gene expression. By coupling with Argonaute family proteins, miRNAs bind to target sites on mRNAs and employ translational repression. A large amount of miRNA-target interactions (MTIs) have been identified by the crosslinking and immunoprecipitation (CLIP) and the photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) along with the next-generation sequencing (NGS). PAR-CLIP shows high efficiency of RNA co-immunoprecipitation, but it also lead to T to C conversion in miRNA-RNA-protein crosslinking regions. This artificial error obviously reduces the mappability of reads. However, a specific tool to analyze CLIP and PAR-CLIP data that takes T to C conversion into account is still in need.ResultsWe herein propose the first CLIP and PAR-CLIP sequencing analysis platform specifically for miRNA target analysis, namely miRTarCLIP. From scratch, it automatically removes adaptor sequences from raw reads, filters low quality reads, reverts C to T, aligns reads to 3'UTRs, scans for read clusters, identifies high confidence miRNA target sites, and provides annotations from external databases. With multi-threading techniques and our novel C to T reversion procedure, miRTarCLIP greatly reduces the running time comparing to conventional approaches. In addition, miRTarCLIP serves with a web-based interface to provide better user experiences in browsing and searching targets of interested miRNAs. To demonstrate the superior functionality of miRTarCLIP, we applied miRTarCLIP to two public available CLIP and PAR-CLIP sequencing datasets. miRTarCLIP not only shows comparable results to that of other existing tools in a much faster speed, but also reveals interesting features among these putative target sites. Specifically, we used miRTarCLIP to disclose that T to C conversion within position 1-7 and that within position 8-14 of miRNA target sites are significantly different (p value = 0.02), and even more significant when focusing on sites targeted by top 102 highly expressed miRNAs only (p value = 0.01). These results comply with previous findings and further suggest that combining miRNA expression and PAR-CLIP data can improve accuracy of the miRNA target prediction.ConclusionTo sum up, we devised a systematic approach for mining miRNA-target sites from CLIP-seq and PAR-CLIP sequencing data, and integrated the workflow with a graphical web-based browser, which provides a user friendly interface and detailed annotations of MTIs. We also showed through real-life examples that miRTarCLIP is a powerful tool for understanding miRNAs. Our integrated tool can be accessed online freely at http://miRTarCLIP.mbc.nctu.edu.tw.

Limited evidence for evolutionarily conserved targeting of long non-coding RNAs by microRNAs.

BackgroundLong non-coding RNAs (lncRNAs) are emerging as important regulators of cell physiology, but it is yet unknown to what extent lncRNAs have evolved to be targeted by microRNAs. Comparative genomics has previously revealed widespread evolutionarily conserved microRNA targeting of protein-coding mRNAs, and here we applied a similar approach to lncRNAs.FindingsWe used a map of putative microRNA target sites in lncRNAs where site conservation was evaluated based on 46 vertebrate species. We compared observed target site frequencies to those obtained with a random model, at variable prediction stringencies. While conserved sites were not present above random expectation in intergenic lncRNAs overall, we observed a marginal over-representation of highly conserved 8-mer sites in a small subset of cytoplasmic lncRNAs (12 sites in 8 lncRNAs at 56% false discovery rate, P = 0.10).ConclusionsEvolutionary conservation in lncRNAs is generally low but patch-wise high, and these patches could, in principle, harbor conserved target sites. However, while our analysis efficiently detected conserved targeting of mRNAs, it provided only limited and marginally significant support for conserved microRNA-lncRNA interactions. We conclude that conserved microRNA-lncRNA interactions could not be reliably detected with our methodology.

Up-Regulated Expression of miR-23a/b Targeted the Pro-Apoptotic Fas in Radiation-Induced thymic Lymphoma

Background: MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs, which usually bind to the 3'-untranslated region of target mRNAs and are capable of inducing posttranscriptional gene regulation by blocking translation or by degrading the target mRNA. However, the expression level of miR-23 in radiation induced carcinogenesis is largely unknown. Methods: Radiation induced thymic lymphoma model in BALB/c mice was set up. miR-23a & miR-23b miRNA levels in different tissues and cells were detected by real-time qPCR. miR-23a/b inhibitor and miR-23a/b mimics were transfected to lymphoma cells and the target of miR-23a/b was identified by microRNA target prediction and Luciferase assays. Results: We found that miR-23a & miR-23b were up-regulated in radiation induced thymic lymphoma tissue samples. Cell death and apoptosis were increased by miR-23a/b inhibitor and decreased by miR-23a/b mimics in lymphoma cells. Computational analysis found a putative target site of miR-23a/b in the 3′UTR of Fas mRNA, which was verified by luciferase reporter assay. Forced over-expression of miR-23a/b decreased the level of Fas protein. Moreover, over-expression of Fas rescued the pro-proliferation effect of miR-23, indicating Fas is a direct mediator of miR-23 functions. Furthermore, contrast to miR-23a/b which was up regulated, the Fas expression level was down-regulated and inversely correlated with miR-23 in split radiation induced lymphoma tissue samples. Finally, our data also indicates that miR-23a could repress Fas much more potent than miR-23b and the additional region besides conserved seed pairing enables miR-23a's higher regulation. Conclusions: In this study, using a radiation induced thymic lymphoma model in BALB/c mice, We conclude that the expression of miR-23a/b is up-regulated in radiation-induced thymic lymphoma and it maybe a novel therapeutic target of that cancer.

Expression of Cereblon (CRBN) Is Associated with Disease Stage, Genetic Subgroups and Specific Micro-RNAs in Multiple Myeloma

AbstractAbstract 1820 Introduction:Since the introduction of thalidomide into the treatment of multiple myeloma (MM) immunomodulatory drugs (IMiDs) have become an essential treatment modality for the management of MM. Recently, cereblon (CRBN) has been identified as the thalidomide binding protein disrupting the E3 ubiquitin ligase complex composed by CRBN, DDB1 and Cul4 (Ito et al., Science 2010). Moreover, CRBN is essential for the antimyeloma activity of lenalidomide and pomalidomide (Zhu et al.; Blood 2011). However, the genetic and epigenetic mechanisms by which CRBN is regulated are not understood so far. Therefore, we aimed to determine if CRBN expression associates with clinically relevant subgroups and response to therapy with lenalidomide. In addition, we investigated additional regulatory layers by identifying key microRNAs (miRNAs) correlated with CRBN expression. Methods:CRBN expression was measured by real-time PCR (qPCR) using CD138 purified plasma cells from 17 patients with a monoclonal gammopathy of undetermined significance (MGUS) and 139 patients with MM. CRBN expression was normalized to GUSB (used as internal control) and to the expression levels of CRBN in normal plasma cells (n=4). MiRNA expression profiles were generated using Agilent mirRNA-arrays on patients with the highest and lowest CRBN mRNA expression levels, respectively (n=42). All patients were characterized by a comprehensive set of FISH probes for the presence of recurring cytogenetic abnormalities. Results:CRBN expression was variable in the investigated samples (median: 0.717; range: 0.078 – 5.285). In patients with MGUS (median: 0.833) CRBN expression was significantly (p=0.01) lower as compared to patients with a MM (median: 0.673). CRBN expression was associated with cytogenetic subgroups (p<0.05), with patients harboring a translocation t(11;14) and gains at 9q34 presenting with the highest CRBN expression levels and patients with a deletion(del) 17p13 or gains at 1q21 with the lowest CRBN expression levels. Out of 526 miRNAs that passed the detection threshold, 28 miRNAs were significantly associated with CRBN expression (p<0.005; >2-fold change). A total of 21 miRNAs were positively correlated and seven miRNAs were negatively correlated with high CRBN expression levels. Of particular interest, miRNAs with conserved target sites in the CRBN 3′-UTR (miRNA -23a and let-7g) were represented in the signature. Summary:The expression of CRBN is variable in plasma cell disorders with decreasing CRBN expression levels in MM compared to the precursor state MGUS and normal plasma cells. Moreover, CRBN is lower expressed in poor cytogenetic subgroups (in particular del17p13 and gains at 1q21). This might provide a novel mechanism underlying the adverse clinic outcome in this patient group. Finally, the differential expression of miRNAs with putative CRBN target sites may uncover an important regulatory mechanism. Disclosures:No relevant conflicts of interest to declare.

Integrative analysis of somatic mutations altering microRNA targeting in cancer genomes.

Determining the functional impact of somatic mutations is crucial to understanding tumorigenesis and metastasis. Recent sequences of several cancers have provided comprehensive lists of somatic mutations across entire genomes, enabling investigation of the functional impact of somatic mutations in non-coding regions. Here, we study somatic mutations in 3′UTRs of genes that have been identified in four cancers and computationally predict how they may alter miRNA targeting, potentially resulting in dysregulation of the expression of the genes harboring these mutations. We find that somatic mutations create or disrupt putative miRNA target sites in the 3′UTRs of many genes, including several genes, such as MITF, EPHA3, TAL1, SCG3, and GSDMA, which have been previously associated with cancer. We also integrate the somatic mutations with germline mutations and results of association studies. Specifically, we identify putative miRNA target sites in the 3′UTRs of BMPR1B, KLK3, and SPRY4 that are disrupted by both somatic and germline mutations and, also, are in linkage disequilibrium blocks with high scoring markers from cancer association studies. The somatic mutation in BMPR1B is located in a target site of miR-125b; germline mutations in this target site have previously been both shown to disrupt regulation of BMPR1B by miR-125b and linked with cancer.

Transcript abundance of the pig stearoyl-CoA desaturase gene has no effect on fatty acid composition in muscle and fat tissues, but its polymorphism within the putative microRNA target site is associated with daily body weight gain and feed conversion ratio1

Fatty acid composition in porcine intramuscular fat affects the dietetic value and technological properties of meat. The stearoyl-CoA desaturase (SCD) gene is a strong positional and functional candidate for fatty acid composition. Our sequence analysis in 4 breeds (Duroc, Pietrain, Polish Landrace, and Polish Large White) revealed a novel SNP in the 5'-flanking sequence and 9 novel SNP and 2 novel indels in the 3' untranslated region (UTR). Transcript level of the SCD in subcutaneous fat was significantly greater than in muscle tissue (n=83; P<0.001) and the interbreed comparison revealed a greater transcript level in the fat tissue of Polish Landrace (P<0.01). We found no association between the abundance of the SCD transcript and fatty acid composition in any of the tissues. We performed an association analysis between 4 SNP (c.-353C>T, c.-233T>C, c.*164A>G, and c.*928G>C), 1 indel (c.*2574_2576delGTC), and production traits in Polish Large White (n=185) and synthetic line 990 (n=243). The most pronounced associations were observed for the c.*928G>C polymorphism, which occurs within a predicted target site for 2 microRNA (ssc-miR-185 and ssc-miR-491). In line 990, this polymorphism was significantly associated with daily BW gain (P<0.04 under the general model) and feed conversion ratio (P<0.0004) but not with fatness traits. The same tendency, but not significant, was observed in the Polish Large White breed. When both breeds were analyzed together, these associations were again highly significant (daily BW gain P<0.003; feed conversion ratio P<0.0001). We conclude that c.*928G>C is a promising marker for both porcine traits.

Role of recently evolved miRNA regulation of sunflower HaWRKY6 in response to temperature damage

MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated. Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing. Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature. Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress.

‘MicroRNA Targets’, a new AthaMap web-tool for genome-wide identification of miRNA targets in Arabidopsis thaliana

BackgroundThe AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation.MethodsTo identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome.ResultsPutative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, ‘MicroRNA Targets’, was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new ‘Small RNA Targets’ web-tool.ConclusionsThe integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/

Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells

The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3'ss, three alternative 5'ss (N4-, N50-, and N62-5'ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5'ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5'ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/β-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5'ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5'ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes.