The Adequate Corpus Luteum: miR-96 Promotes Luteal Cell Survival and Progesterone Production.

PubMed

Mohammed, Bushra T; Sontakke, Sadanand D; Ioannidis, Jason; Duncan, W Colin; Donadeu, F Xavier

2017-07-01

Inadequate progesterone production from the corpus luteum is associated with pregnancy loss. Data available in model species suggest important roles of microRNAs (miRNAs) in luteal development and maintenance. To comprehensively investigate the involvement of miRNAs during the ovarian follicle-luteal transition. The effects of specific miRNAs on survival and steroid production by human luteinized granulosa cells (hLGCs) were tested using specific miRNA inhibitors. Candidate miRNAs were identified through microarray analyses of follicular and luteal tissues in a bovine model. An academic institution in the United Kingdom associated with a teaching hospital. hLGCs were obtained by standard transvaginal follicular-fluid aspiration from 35 women undergoing assisted conception. Inhibition of candidate miRNAs in vitro. Levels of miRNAs, mRNAs, FOXO1 protein, apoptosis, and steroids were measured in tissues and/or cultured cells. Two specific miRNA clusters, miR-183-96-182 and miR-212-132, were dramatically increased in luteal relative to follicular tissues. miR-96 and miR-132 were the most upregulated miRNAs within each cluster. Database analyses identified FOXO1 as a putative target of both these miRNAs. In cultured hLGCs, inhibition of miR-96 increased apoptosis and FOXO1 protein levels, and decreased progesterone production. These effects were prevented by small interfering RNA-mediated downregulation of FOXO1. In bovine luteal cells, miR-96 inhibition also led to increases in apoptosis and FOXO1 protein levels. miR-96 targets FOXO1 to regulate luteal development through effects on cell survival and steroid production. The miR-183-96-182 cluster could provide a novel target for the manipulation of luteal function. Copyright © 2017 Endocrine Society

Blood-Bourne MicroRNA Biomarker Evaluation in Attention-Deficit/Hyperactivity Disorder of Han Chinese Individuals: An Exploratory Study.

PubMed

Wang, Liang-Jen; Li, Sung-Chou; Lee, Min-Jing; Chou, Miao-Chun; Chou, Wen-Jiun; Lee, Sheng-Yu; Hsu, Chih-Wei; Huang, Lien-Hung; Kuo, Ho-Chang

2018-01-01

Background: Attention-deficit/hyperactivity disorder (ADHD) is a highly genetic neurodevelopmental disorder, and its dysregulation of gene expression involves microRNAs (miRNAs). The purpose of this study was to identify potential miRNAs biomarkers and then use these biomarkers to establish a diagnostic panel for ADHD. Design and methods: RNA samples from white blood cells (WBCs) of five ADHD patients and five healthy controls were combined to create one pooled patient library and one control library. We identified 20 candidate miRNAs with the next-generation sequencing (NGS) technique (Illumina). Blood samples were then collected from a Training Set (68 patients and 54 controls) and a Testing Set (20 patients and 20 controls) to identify the expression profiles of these miRNAs with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used receiver operating characteristic (ROC) curves and the area under the curve (AUC) to evaluate both the specificity and sensitivity of the probability score yielded by the support vector machine (SVM) model. Results: We identified 13 miRNAs as potential ADHD biomarkers. The ΔCt values of these miRNAs in the Training Set were integrated to create a biomarker model using the SVM algorithm, which demonstrated good validity in differentiating ADHD patients from control subjects (sensitivity: 86.8%, specificity: 88.9%, AUC: 0.94, p < 0.001). The results of the blind testing showed that 85% of the subjects in the Testing Set were correctly classified using the SVM model alignment (AUC: 0.91, p < 0.001). The discriminative validity is not influenced by patients' age or gender, indicating both the robustness and the reliability of the SVM classification model. Conclusion: As measured in peripheral blood, miRNA-based biomarkers can aid in the differentiation of ADHD in clinical settings. Additional studies are needed in the future to clarify the ADHD-associated gene functions and biological mechanisms modulated by miRNAs.

Rapid upregulation and clearance of distinct circulating microRNAs after prolonged aerobic exercise

PubMed Central

Park, Joseph; Min, Pil-Ki; Isaacs, Stephanie; Parker, Beth A.; Thompson, Paul D.; Troyanos, Chris; D'Hemecourt, Pierre; Dyer, Sophia; Thiel, Marissa; Hale, Andrew; Chan, Stephen Y.

2014-01-01

Short nonprotein coding RNA molecules, known as microRNAs (miRNAs), are intracellular mediators of adaptive processes, including muscle hypertrophy, contractile force generation, and inflammation. During basal conditions and tissue injury, miRNAs are released into the bloodstream as “circulating” miRNAs (c-miRNAs). To date, the impact of extended-duration, submaximal aerobic exercise on plasma concentrations of c-miRNAs remains incompletely characterized. We hypothesized that specific c-miRNAs are differentially upregulated following prolonged aerobic exercise. To test this hypothesis, we measured concentrations of c-miRNAs enriched in muscle (miR-1, miR-133a, miR-499–5p), cardiac tissue (miR-208a), and the vascular endothelium (miR-126), as well as those important in inflammation (miR-146a) in healthy male marathon runners (N = 21) at rest, immediately after a marathon (42-km foot race), and 24 h after the race. In addition, we compared c-miRNA profiles to those of conventional protein biomarkers reflective of skeletal muscle damage, cardiac stress and necrosis, and systemic inflammation. Candidate c-miRNAs increased immediately after the marathon and declined to prerace levels or lower after 24 h of race completion. However, the magnitude of change for each c-miRNA differed, even when originating from the same tissue type. In contrast, traditional biomarkers increased after exercise but remained elevated 24 h postexercise. Thus c-miRNAs respond differentially to prolonged exercise, suggesting the existence of specific mechanisms of c-miRNA release and clearance not fully explained by generalized cellular injury. Furthermore, c-miRNA expression patterns differ in a temporal fashion from corollary conventional tissue-specific biomarkers, emphasizing the potential of c-miRNAs as unique, real-time markers of exercise-induced tissue adaptation. PMID:24436293

Induction of interferon lambda in influenza a virus infected cells treated with shRNAs against M1 transcript.

PubMed

Švančarová, P; Svetlíková, D; Betáková, T

2015-06-01

RNA interference (RNAi) represents a form of post-transcriptional gene silencing mediated by small interfering RNAs (siRNA) and provides a powerful tool to specifically inhibit viral infection. To investigate therapeutic capacity of siRNAs targeting M gene, six vectors with U1-short hairpin RNA (shRNA) expression system were prepared and tested in infected cells and animals. In infected cells, three of six shRNAs targeting M1 gene significantly (P <0,01) reduced the virus titer to 66%, 45% or 21%, respectively. Replication of IAV and levels of M1 RNAs were significantly reduced in the cells transfected with shRNAs, which decreased the virus titer. IFN-α/β altered in shRNAs-treated cells. The level of IFN-λ (type III interferon) mRNA was significantly increased in the infected cells treated with shM22, shM349, shM522, and (type I interferon) as well as IP-10 (type II interferon) mRNAs were not significantly their mixtures. The increased level of IFN-λ mRNA corresponded to significantly increased level of RIG-1 mRNA. shRNAs inhibited influenza virus infection in a gene-specific manner in co-operation with IFN-λ. Some constructs targeting the M1 transcript prolonged the survival of infected mice.

MicroRNAs and toxicology: A love marriage.

PubMed

Schraml, Elisabeth; Hackl, Matthias; Grillari, Johannes

2017-01-01

With the dawn of personalized medicine, secreted microRNAs (miRNAs) have come into the very focus of biomarker development for various diseases. MiRNAs fulfil key requirements of diagnostic tools such as i) non or minimally invasive accessibility, ii) robust, standardized and non-expensive quantitative analysis, iii) rapid turnaround of the test result and iv) most importantly because they provide a comprehensive snapshot of the ongoing physiologic processes in cells and tissues that package and release miRNAs into cell-free space. These characteristics have also established circulating miRNAs as promising biomarker candidates for toxicological studies, where they are used as biomarkers of drug-, or chemical-induced tissue injury for safety assessment. The tissue-specificity and early release of circulating miRNAs upon tissue injury, when damage is still reversible, are main factors for their clinical utility in toxicology. Here we summarize in brief, current knowledge of this field.

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona.

PubMed

Gandhi, Shashank; Haeussler, Maximilian; Razy-Krajka, Florian; Christiaen, Lionel; Stolfi, Alberto

2017-05-01

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome organization and characteristics of soybean microRNAs

PubMed Central

2012-01-01

Background microRNAs (miRNAs) are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s) of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum-inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs) and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets. Results We identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean. Conclusions Genome organization of soybean miRNAs suggests that they are actively evolving. Distinct family characteristics of soybean miRNAs suggest continuous diversification of function. Inverse organ-specific expression between selected miRNAs and their targets in the roots and nodules, suggested a potential role for these miRNAs in regulating nodule development. PMID:22559273

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

PubMed Central

Raabe, Carsten A.; Sanchez, Cecilia P.; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V.; Chinni, Suresh V.; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y.; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S.

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors. PMID:19864253

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum.

PubMed

Raabe, Carsten A; Sanchez, Cecilia P; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V; Chinni, Suresh V; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Conservation of small RNA pathways in platypus

PubMed Central

Murchison, Elizabeth P.; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J.

2008-01-01

Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense. PMID:18463306

Conservation of small RNA pathways in platypus.

PubMed

Murchison, Elizabeth P; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J

2008-06-01

Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense.

Screening and confirmation of microRNA markers for forensic body fluid identification.

PubMed

Wang, Zheng; Zhang, Ji; Luo, Haibo; Ye, Yi; Yan, Jing; Hou, Yiping

2013-01-01

MicroRNAs (miRNAs, ∼22 nucleotides) are small, non-protein coding RNAs that regulate gene expression at the post-transcriptional level. MiRNAs can express in a tissue-specific manner, and have been introduced to forensic body fluid identification. In this study, we employed the qPCR-array (TaqMan(®) Array Human MicroRNA Cards) to screen the body fluid-specific miRNAs. Seven candidate miRNAs were identified as potentially body fluid-specific and could be used as forensically relevant body fluid markers: miR16 and miR486 for venous blood, miR888 and miR891a for semen, miR214 for menstrual blood, miR124a for vaginal secretions, and miR138-2 for saliva. The candidate miRNA markers were then validated via hydrolysis probes quantitative real-time polymerase chain reaction (TaqMan-qPCR). In addition, BestKeeper software was used to validate the expression stability of four genes, RNU44, RNU48, U6 and U6b, regularly used as reference genes (RGs) for studies involving forensic body fluids. The current study suggests that U6 could be used as a proper RG of miRNAs in forensic body fluid identification. The relative expression ratios (R) of miR486, miR888, miR214, miR16 and miR891a can differentiate the target body fluid from other body fluids that were tested in this study. The detection limit of TaqMan-qPCR of the five confirmed miRNA markers was 10pg of total RNA. The effect of time-wise degradation of blood stains and semen stains for 1 month under normal laboratory conditions was tested and did not significantly affect the detection results. Herein, this study proposes five body fluid-specific miRNAs for the forensic identification of venous blood, semen, and menstrual blood, of which miR486, miR888, and miR214 may be used as new markers for body fluid identification. Additional work remains necessary in search for suitable miRNA markers and stable RGs for forensic body fluid identification. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

PubMed Central

Kang, Shih-Ting; Hsieh, Yi-Shan; Feng, Chi-Ting; Chen, Yu-Ting; Yang, Pok Eric; Chen, Wei-Ming

2018-01-01

MicroRNAs (miRNAs) are 18–25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs’ short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences’ platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers. PMID:29208706

Electrochemical determination of microRNAs based on isothermal strand-displacement polymerase reaction coupled with multienzyme functionalized magnetic micro-carriers.

PubMed

Ma, Wen; Situ, Bo; Lv, Weifeng; Li, Bo; Yin, Xiaomao; Vadgama, Pankaj; Zheng, Lei; Wang, Wen

2016-06-15

MicroRNAs (miRNAs) show great potential for disease diagnostics due to their specific molecular profiles. Detection of miRNAs remains challenging and often requires sophisticated platforms. Here we report a multienzyme-functionalized magnetic microcarriers-assisted isothermal strand-displacement polymerase reaction (ISDPR) for quantitative detection of miRNAs. Magnetic micro-carriers (MMCs) were functionalized with molecular beacons to enable miRNAs recognition and magnetic separation. The target miRNAs triggered a phi29-mediated ISDPR, which can produce biotin-modified sequences on the MMCs. Streptavidin-alkaline phosphatase was then conjugated to the MMC surface through biotin-streptavidin interactions. In the presence of 2-phospho-L-ascorbic acid, miRNAs were quantitatively determined on a screen-printed carbon electrode from the anodic current of the enzymatic product. We show that this method enables detection of miRNAs as low as 9 fM and allows the discrimination of one base mismatched sequence. The proposed method was also successfully applied to analyze miRNAs in clinical tumor samples. This paper reports a new strategy for miRNAs analysis with high sensitivity, simplicity, and low cost. It would be particularly useful for rapid point-of-care testing of miRNAs in clinical laboratory. Copyright © 2015 Elsevier B.V. All rights reserved.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

PubMed

Ying, Na; Ju, Chuanjing; Sun, Xiuwei; Li, Letian; Chang, Hongbiao; Song, Guangping; Li, Zhongyi; Wan, Jiayu; Dai, Enyong

2017-01-01

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Adaptive evolution and functional innovation of Populus-specific recently evolved microRNAs.

PubMed

Xie, Jianbo; Yang, Xiaohui; Song, Yuepeng; Du, Qingzhang; Li, Ying; Chen, Jinhui; Zhang, Deqiang

2017-01-01

Lineage-specific microRNAs (miRNAs) undergo rapid turnover during evolution; however, their origin and functional importance have remained controversial. Here, we examine the origin, evolution, and potential roles in local adaptation of Populus-specific miRNAs, which originated after the recent salicoid-specific, whole-genome duplication. RNA sequencing was used to generate extensive, comparable miRNA and gene expression data for six tissues. A natural population of Populus trichocarpa and closely related species were used to study the divergence rates, evolution, and adaptive variation of miRNAs. MiRNAs that originated in 5' untranslated regions had higher expression levels and their expression showed high correlation with their host genes. Compared with conserved miRNAs, a significantly higher proportion of Populus-specific miRNAs appear to target genes that were duplicated in salicoids. Examination of single nucleotide polymorphisms in Populus-specific miRNA precursors showed high amounts of population differentiation. We also characterized the newly emerged MIR6445 family, which could trigger the production of phased small interfering RNAs from NAC mRNAs, which encode a transcription factor with primary roles in a variety of plant developmental processes. Together, these observations provide evolutionary insights into the birth and potential roles of Populus-specific miRNAs in genome maintenance, local adaptation, and functional innovation. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Genome-Wide Discovery of Long Non-Coding RNAs in Rainbow Trout.

PubMed

Al-Tobasei, Rafet; Paneru, Bam; Salem, Mohamed

2016-01-01

The ENCODE project revealed that ~70% of the human genome is transcribed. While only 1-2% of the RNAs encode for proteins, the rest are non-coding RNAs. Long non-coding RNAs (lncRNAs) form a diverse class of non-coding RNAs that are longer than 200 nt. Emerging evidence indicates that lncRNAs play critical roles in various cellular processes including regulation of gene expression. LncRNAs show low levels of gene expression and sequence conservation, which make their computational identification in genomes difficult. In this study, more than two billion Illumina sequence reads were mapped to the genome reference using the TopHat and Cufflinks software. Transcripts shorter than 200 nt, with more than 83-100 amino acids ORF, or with significant homologies to the NCBI nr-protein database were removed. In addition, a computational pipeline was used to filter the remaining transcripts based on a protein-coding-score test. Depending on the filtering stringency conditions, between 31,195 and 54,503 lncRNAs were identified, with only 421 matching known lncRNAs in other species. A digital gene expression atlas revealed 2,935 tissue-specific and 3,269 ubiquitously-expressed lncRNAs. This study annotates the lncRNA rainbow trout genome and provides a valuable resource for functional genomics research in salmonids.

Literature-based condition-specific miRNA-mRNA target prediction.

PubMed

Oh, Minsik; Rhee, Sungmin; Moon, Ji Hwan; Chae, Heejoon; Lee, Sunwon; Kang, Jaewoo; Kim, Sun

2017-01-01

miRNAs are small non-coding RNAs that regulate gene expression by binding to the 3'-UTR of genes. Many recent studies have reported that miRNAs play important biological roles by regulating specific mRNAs or genes. Many sequence-based target prediction algorithms have been developed to predict miRNA targets. However, these methods are not designed for condition-specific target predictions and produce many false positives; thus, expression-based target prediction algorithms have been developed for condition-specific target predictions. A typical strategy to utilize expression data is to leverage the negative control roles of miRNAs on genes. To control false positives, a stringent cutoff value is typically set, but in this case, these methods tend to reject many true target relationships, i.e., false negatives. To overcome these limitations, additional information should be utilized. The literature is probably the best resource that we can utilize. Recent literature mining systems compile millions of articles with experiments designed for specific biological questions, and the systems provide a function to search for specific information. To utilize the literature information, we used a literature mining system, BEST, that automatically extracts information from the literature in PubMed and that allows the user to perform searches of the literature with any English words. By integrating omics data analysis methods and BEST, we developed Context-MMIA, a miRNA-mRNA target prediction method that combines expression data analysis results and the literature information extracted based on the user-specified context. In the pathway enrichment analysis using genes included in the top 200 miRNA-targets, Context-MMIA outperformed the four existing target prediction methods that we tested. In another test on whether prediction methods can re-produce experimentally validated target relationships, Context-MMIA outperformed the four existing target prediction methods. In summary, Context-MMIA allows the user to specify a context of the experimental data to predict miRNA targets, and we believe that Context-MMIA is very useful for predicting condition-specific miRNA targets.

A Panel of MicroRNAs as Diagnostic Biomarkers for the Identification of Prostate Cancer.

PubMed

Daniel, Rhonda; Wu, Qianni; Williams, Vernell; Clark, Gene; Guruli, Georgi; Zehner, Zendra

2017-06-16

Prostate cancer is the most common non-cutaneous cancer among men; yet, current diagnostic methods are insufficient, and more reliable diagnostic markers need to be developed. One answer that can bridge this gap may lie in microRNAs. These small RNA molecules impact protein expression at the translational level, regulating important cellular pathways, the dysregulation of which can exert tumorigenic effects contributing to cancer. In this study, high throughput sequencing of small RNAs extracted from blood from 28 prostate cancer patients at initial stages of diagnosis and prior to treatment was used to identify microRNAs that could be utilized as diagnostic biomarkers for prostate cancer compared to 12 healthy controls. In addition, a group of four microRNAs (miR-1468-3p, miR-146a-5p, miR-1538 and miR-197-3p) was identified as normalization standards for subsequent qRT-PCR confirmation. qRT-PCR analysis corroborated microRNA sequencing results for the seven top dysregulated microRNAs. The abundance of four microRNAs (miR-127-3p, miR-204-5p, miR-329-3p and miR-487b-3p) was upregulated in blood, whereas the levels of three microRNAs (miR-32-5p, miR-20a-5p and miR-454-3p) were downregulated. Data analysis of the receiver operating curves for these selected microRNAs exhibited a better correlation with prostate cancer than PSA (prostate-specific antigen), the current gold standard for prostate cancer detection. In summary, a panel of seven microRNAs is proposed, many of which have prostate-specific targets, which may represent a significant improvement over current testing methods.

MicroRNA-Regulated Non-Viral Vectors with Improved Tumor Specificity in an Orthotopic Rat Model of Hepatocellular Carcinoma

PubMed Central

Ronald, John A.; Katzenberg, Regina; Nielsen, Carsten H.; Jae, Hwan Jun; Hofmann, Lawrence V.; Gambhir, Sanjiv S.

2013-01-01

In hepatocellular carcinoma, tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs are powerful negative regulators of gene expression and many are down-regulated in human HCC. We identified seven miRNAs that are also down-regulated in tumors in a rat hepatoma model (p<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these microRNAs. Attenuation of luciferase expression by the corresponding microRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two microRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared to control vector (6.40% and 0.26%, respectively; p<0.05). In tumor, miR-122 had a non-significant trend towards decreased (~50%) expression , while miR-26 had no significant effect on tumor expression. To our knowledge this is the first work using differentially expressed microRNAs to de-target transgene expression in an orthotopic hepatoma model and identification of miR-26a in addition to miR-122 for de-targeting liver. Considering the heterogeneity of microRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease. PMID:23719066

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

PubMed Central

Kang, Kang; Zhang, Xiaoying; Liu, Hongtao; Wang, Zhiwei; Zhong, Jiasheng; Huang, Zhenting; Peng, Xiao; Zeng, Yan; Wang, Yuna; Yang, Yi; Luo, Jun; Gou, Deming

2012-01-01

Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers. PMID:23152780

USDA Potato Small RNA Database

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNAs) are now understood to be involved in gene regulation, function and development. High throughput sequencing (HTS) of sRNAs generates large data sets for analyzing the abundance, source and roles for specific sRNAs. These sRNAs result from transcript degradation as well as specific ...

Development of novel cardiovascular therapeutics from small regulatory RNA molecules--an outline of key requirements.

PubMed

Poller, W; Fechner, H

2010-01-01

Understanding of the roles of RNAs within the cell has changed and expanded dramatically during the past few years. Based on fundamentally new insights it is now increasingly possible to employ RNAs as highly valuable tools in molecular biology and medicine. At present, the most important therapeutic strategies are based on non-coding regulatory RNAs inducing RNA interference (RNAi) to silence single genes, and on modulation of cellular microRNAs (miRNAs) to alter complex gene expression patterns in diseased organs. Only recently it became possible to target therapeutic RNAi to specific organs via organotropic viral vector systems and we discuss the most recent strategies in this field, e.g. heart failure treatment by cardiac-targeted RNAi. Due to the peculiar biochemical properties of small RNA molecules, true therapeutic translation of results in vitro is more demanding than with small molecule drugs or proteins. Specifically, there is a critical requirement for extensive studies in animal models of human disease after pre-testing of the RNAi tools in vitro. This requirement likewise applies for miRNA modulations which have complex consequences in the recipient dependent on biochemical stability and distribution of the therapeutic RNA. Problems not yet fully solved are the prediction of targets and specificity of the RNA tools. However, major progress has been made to achieve their tissue-specific and regulatable expression, and breakthroughs in vector technologies from the gene therapy field have fundamentally improved safety and efficacy of RNA-based therapeutic approaches, too. In summary, insight into the molecular mechanisms of action of regulatory RNAs in combination with new delivery tools for RNA therapeutics will significantly expand our cardiovascular therapeutic repertoire beyond classical pharmacology.

Global microRNA expression profiling of microdissected tissues identifies miR-135b as a novel biomarker for pancreatic ductal adenocarcinoma.

PubMed

Munding, Johanna B; Adai, Alex T; Maghnouj, Abdelouahid; Urbanik, Aleksandra; Zöllner, Hannah; Liffers, Sven T; Chromik, Ansgar M; Uhl, Waldemar; Szafranska-Schwarzbach, Anna E; Tannapfel, Andrea; Hahn, Stephan A

2012-07-15

Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI=(80.5, 98.5)] and 93.4% [95% CI=(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC. Copyright © 2011 UICC.

Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice

PubMed Central

Koval, Erica D.; Shaner, Carey; Zhang, Peter; du Maine, Xavier; Fischer, Kimberlee; Tay, Jia; Chau, B. Nelson; Wu, Gregory F.; Miller, Timothy M.

2013-01-01

microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1)G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS. PMID:23740943

A Customized Quantitative PCR MicroRNA Panel Provides a Technically Robust Context for Studying Neurodegenerative Disease Biomarkers and Indicates a High Correlation Between Cerebrospinal Fluid and Choroid Plexus MicroRNA Expression.

PubMed

Wang, Wang-Xia; Fardo, David W; Jicha, Gregory A; Nelson, Peter T

2017-12-01

MicroRNA (miRNA) expression varies in association with different tissue types and in diseases. Having been found in body fluids including blood and cerebrospinal fluid (CSF), miRNAs constitute potential biomarkers. CSF miRNAs have been proposed as biomarkers for neurodegenerative diseases; however, there is a lack of consensus about the best candidate miRNA biomarkers and there has been variability in results from different research centers, perhaps due to technical factors. Here, we sought to optimize technical parameters for CSF miRNA studies. We examined different RNA isolation methods and performed miRNA expression profiling with TaqMan® miRNA Arrays. More specifically, we developed a customized CSF-miRNA low-density array (TLDA) panel that contains 47 targets: miRNAs shown previously to be relevant to neurodegenerative disease, miRNAs that are abundant in CSF, data normalizers, and controls for potential blood and tissue contamination. The advantages of using this CSF-miRNA TLDA panel include specificity, sensitivity, fast processing and data analysis, and cost effectiveness. We optimized technical parameters for this assay. Further, the TLDA panel can be tailored to other specific purposes. We tested whether the profile of miRNAs in the CSF resembled miRNAs isolated from brain tissue (hippocampus or cerebellum), blood, or the choroid plexus. We found that the CSF miRNA expression profile most closely resembles that of choroid plexus tissue, underscoring the potential importance of choroid plexus-derived signaling through CSF miRNAs. In summary, the TLDA miRNA array panel will enable evaluation and discovery of CSF miRNA biomarkers and can potentially be utilized in clinical diagnosis and disease stage monitoring.

Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity

PubMed Central

Rizzo, Milena; Evangelista, Monica; Simili, Marcella; Mariani, Laura; Pitto, Letizia; Rainaldi, Giuseppe

2011-01-01

The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity. PMID:21765199

Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity.

PubMed

Rizzo, Milena; Evangelista, Monica; Simili, Marcella; Mariani, Laura; Pitto, Letizia; Rainaldi, Giuseppe

2011-07-01

The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity.

Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis.

PubMed

Meningher, Tal; Lerman, Galya; Regev-Rudzki, Neta; Gold, Daniel; Ben-Dov, Iddo Z; Sidi, Yechezkel; Avni, Dror; Schwartz, Eli

2017-02-01

Schistosomiasis traditionally has been diagnosed by detecting eggs in stool or urine. However, the sensitivity of these examinations is limited, especially in travelers with a low worm burden. Serologic tests have a greater sensitivity, but their results remain positive regardless of treatment and thus cannot be used for follow-up of patients. We hypothesized that detection of worm microRNAs (miRNAs) in serum can overcome the drawbacks of the existing diagnostic methods. Twenty-six returning travelers with schistosomiasis (based on positive results of serologic tests or detection of ova) and 17 healthy controls were included in the study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification of miRNA extracted directly from 500 µL of serum had limited sensitivity and specificity. However, qRT-PCR analysis of RNA extracted from 200 μL of serum extracellular vesicles detected 4 schistosomal miRNAs; the sensitivity and specificity of the 2 highest expressed miRNAs (bantam and miR-2c-3p) were 86% and 84%, respectively. In 7 patients with posttreatment serum available for analysis, we observed outcomes ranging from a reduction in the schistosomal miRNA level to full recovery from disease. qRT-PCR of pathogen miRNAs isolated from extracellular vesicles in sera from infected individuals may provide a new tool for diagnosing schistosomiasis in patients with a low parasite burden. This assay could also be used for evaluating the outcome of therapy, as well as disease-control programs. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Digital genome-wide ncRNA expression, including SnoRNAs, across 11 human tissues using polyA-neutral amplification.

PubMed

Castle, John C; Armour, Christopher D; Löwer, Martin; Haynor, David; Biery, Matthew; Bouzek, Heather; Chen, Ronghua; Jackson, Stuart; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

2010-07-26

Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap.

PubMed

Varkonyi-Gasic, Erika; Gould, Nick; Sandanayaka, Manoharie; Sutherland, Paul; MacDiarmid, Robin M

2010-08-04

Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants.

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap

PubMed Central

2010-01-01

Background Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Results Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. Conclusions This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants. PMID:20682080

On BC1 RNA and the fragile X mental retardation protein

PubMed Central

Iacoangeli, Anna; Rozhdestvensky, Timofey S.; Dolzhanskaya, Natalia; Tournier, Barthélémy; Schütt, Janin; Brosius, Jürgen; Denman, Robert B.; Khandjian, Edouard W.; Kindler, Stefan; Tiedge, Henri

2008-01-01

The fragile X mental retardation protein (FMRP), the functional absence of which causes fragile X syndrome, is an RNA-binding protein that has been implicated in the regulation of local protein synthesis at the synapse. The mechanism of FMRP's interaction with its target mRNAs, however, has remained controversial. In one model, it has been proposed that BC1 RNA, a small non-protein-coding RNA that localizes to synaptodendritic domains, operates as a requisite adaptor by specifically binding to both FMRP and, via direct base-pairing, to FMRP target mRNAs. Other models posit that FMRP interacts with its target mRNAs directly, i.e., in a BC1-independent manner. Here five laboratories independently set out to test the BC1–FMRP model. We report that specific BC1–FMRP interactions could be documented neither in vitro nor in vivo. Interactions between BC1 RNA and FMRP target mRNAs were determined to be of a nonspecific nature. Significantly, the association of FMRP with bona fide target mRNAs was independent of the presence of BC1 RNA in vivo. The combined experimental evidence is discordant with a proposed scenario in which BC1 RNA acts as a bridge between FMRP and its target mRNAs and rather supports a model in which BC1 RNA and FMRP are translational repressors that operate independently. PMID:18184799

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

PubMed Central

Li, Dandan; Wang, Yanhong; Zhang, Kun; Jiao, Zhujin; Zhu, Xiaopeng; Skogerboe, Geir; Guo, Xiangqian; Chinnusamy, Viswanathan; Bi, Lijun; Huang, Yongping; Dong, Shuanglin; Chen, Runsheng; Kan, Yunchao

2011-01-01

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50–500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2′-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution. PMID:21227919

Circulating RNAs as new biomarkers for detecting pancreatic cancer

PubMed Central

Kishikawa, Takahiro; Otsuka, Motoyuki; Ohno, Motoko; Yoshikawa, Takeshi; Takata, Akemi; Koike, Kazuhiko

2015-01-01

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers. PMID:26229396

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

PubMed

Friedländer, Marc R; Mackowiak, Sebastian D; Li, Na; Chen, Wei; Rajewsky, Nikolaus

2012-01-01

microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically downregulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers.

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

PubMed Central

Caplen, Natasha J.; Parrish, Susan; Imani, Farhad; Fire, Andrew; Morgan, Richard A.

2001-01-01

Short interfering RNAs (siRNAs) are double-stranded RNAs of ≈21–25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5′-phosphate/3′-hydroxyl ends and a 2-base 3′ overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells. PMID:11481446

Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carbonell, Alberto; Martinez de Alba, Angel-Emilio; Flores, Ricardo

2008-02-05

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one ofmore » the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.« less

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

PubMed Central

Kasai, Atsushi; Sano, Teruo; Harada, Takeo

2013-01-01

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock. PMID:23469061

Identification of host miRNAs that may limit human rhinovirus replication

PubMed Central

Bondanese, Victor Paky; Francisco-Garcia, Ana; Bedke, Nicole; Davies, Donna E; Sanchez-Elsner, Tilman

2014-01-01

AIM: To test whether the replication of human rhinovirus (HRV) is regulated by microRNAs in human bronchial epithelial cells. METHODS: For the present study, the human cell line BEAS-2B (derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by siRNA transfection in BEAS-2B cells, was performed in order to inhibit microRNA maturation globally. Alternatively, antisense oligonucleotides (anti-miRs) were transfected to inhibit the activity of specific microRNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Association between microRNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-qPCR. Targetscan v.6 was used to predict microRNA target sites on several HRV strains. RESULTS: Here, we show that microRNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature microRNAs in a bronchial epithelial cell line (BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for microRNA activity suggesting that microRNAs bind to viral RNA during infection. In order to identify specific microRNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of microRNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses (HRV-1B, -16, -14, -27). Our results suggest that, out of this group of microRNAs, miR-128 and miR-155 contribute to the innate defense against HRV-1B: transfection of specific anti-miRs increased viral replication, as anticipated in-silico. CONCLUSION: Taken together, our results suggest that pathological changes in microRNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virus-induced exacerbations. PMID:25426267

Dynamic Organization of lncRNA and Circular RNA Regulators Collectively Controlled Cardiac Differentiation in Humans.

PubMed

Li, Yongsheng; Zhang, Jinwen; Huo, Caiqin; Ding, Na; Li, Junyi; Xiao, Jun; Lin, Xiaoyu; Cai, Benzhi; Zhang, Yunpeng; Xu, Juan

2017-10-01

Advances in developmental cardiology have increased our understanding of the early aspects of heart differentiation. However, understanding noncoding RNA (ncRNA) transcription and regulation during this process remains elusive. Here, we constructed transcriptomes for both long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in four important developmental stages ranging from early embryonic to cardiomyocyte based on high-throughput sequencing datasets, which indicate the high stage-specific expression patterns of two ncRNA types. Additionally, higher similarities of samples within each stage were found, highlighting the divergence of samples collected from distinct cardiac developmental stages. Next, we developed a method to identify numerous lncRNA and circRNA regulators whose expression was significantly stage-specific and shifted gradually and continuously during heart differentiation. We inferred that these ncRNAs are important for the stages of cardiac differentiation. Moreover, transcriptional regulation analysis revealed that the expression of stage-specific lncRNAs is controlled by known key stage-specific transcription factors (TFs). In addition, circRNAs exhibited dynamic expression patterns independent from their host genes. Functional enrichment analysis revealed that lncRNAs and circRNAs play critical roles in pathways that are activated specifically during heart differentiation. We further identified candidate TF-ncRNA-gene network modules for each differentiation stage, suggesting the dynamic organization of lncRNAs and circRNAs collectively controlled cardiac differentiation, which may cause heart-related diseases when defective. Our study provides a foundation for understanding the dynamic regulation of ncRNA transcriptomes during heart differentiation and identifies the dynamic organization of novel key lncRNAs and circRNAs to collectively control cardiac differentiation. Copyright © 2017. Published by Elsevier B.V.

Digital Genome-Wide ncRNA Expression, Including SnoRNAs, across 11 Human Tissues Using PolyA-Neutral Amplification

PubMed Central

Castle, John C.; Armour, Christopher D.; Löwer, Martin; Haynor, David; Biery, Matthew; Bouzek, Heather; Chen, Ronghua; Jackson, Stuart; Johnson, Jason M.; Rohl, Carol A.; Raymond, Christopher K.

2010-01-01

Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available. PMID:20668672

The miRNA Mirage: How Close Are We to Finding a Non-Invasive Diagnostic Biomarker in Endometriosis? A Systematic Review

PubMed Central

Agrawal, Swati; Tapmeier, Thomas T.; Rahmioglu, Nilufer; Kirtley, Shona; Zondervan, Krina T.; Becker, Christian M.

2018-01-01

Background: Endometriosis is a common disorder of the reproductive age group, characterised by the presence of ectopic endometrial tissue. The disease not only causes enormous suffering to the affected women, but also brings a tremendous medical and economic burden to bear on society. There is a long lag phase between the onset and diagnosis of the disease, mainly due to its non-specific symptoms and the lack of a non-invasive test. Endometriosis can only be diagnosed invasively by laparoscopy. A specific, non-invasive test to diagnose endometriosis is an unmet clinical need. The recent discovery of microRNAs (miRNAs) as modulators of gene expression, and their stability and specificity, make them an attractive candidate biomarker. Various studies on miRNAs in endometriosis have identified their cardinal role in the pathogenesis of the disease, and have proposed them as potential biomarkers in endometriosis. Rationale/Objectives: The aims of this review were to study the role of circulatory miRNAs in endometriosis, and bring to light whether circulatory miRNAs could be potential non-invasive biomarkers to diagnose the disease. Search methods: Three databases, PubMed, EMBASE, and BIOSIS were searched, using a combination of Mesh or Emtree headings and free-text terms, to identify literature relating to circulating miRNAs in endometriosis published from 1996 to 31 December 2017. Only peer-reviewed, full-text original research articles in English were included in the current review. The studies meeting the inclusion criteria were critically assessed and checked using the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool. The dysregulated miRNAs were assessed regarding the concordance between the various studies and their role in the disease. Outcomes: Nine studies were critically analysed, and 42 different miRNAs were found to be dysregulated in them, with only one common miRNA (miR-20a) differentially expressed in more than one study. miR-17-5p/20a, miR-200, miR-199a, miR-143, and miR-145 were explored for their pivotal role in the aetiopathogenesis of endometriosis. Wider implications: It is emerging that miRNAs play a central role in the pathogenesis of endometriosis and have the potential of being promising biomarkers. Circulating miRNAs as a non-invasive diagnostic tool may shorten the delay in the diagnosis of the disease, thus alleviating the suffering of women and reducing the burden on health care systems. However, despite numerous studies on circulating miRNAs in endometriosis, no single miRNA or any panel of them seems to meet the criteria of a diagnostic biomarker. The disagreement between the various studies upholds the demand of larger, well-controlled systematic validation studies with uniformity in the research approaches and involving diverse populations. PMID:29463003

Multilevel regulation of gene expression by microRNAs.

PubMed

Makeyev, Eugene V; Maniatis, Tom

2008-03-28

MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

PubMed Central

Bramsen, Jesper B.; Pakula, Malgorzata M.; Hansen, Thomas B.; Bus, Claus; Langkjær, Niels; Odadzic, Dalibor; Smicius, Romualdas; Wengel, Suzy L.; Chattopadhyaya, Jyoti; Engels, Joachim W.; Herdewijn, Piet; Wengel, Jesper; Kjems, Jørgen

2010-01-01

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low. PMID:20453030

Guide-substrate base-pairing requirement for box H/ACA RNA-guided RNA pseudouridylation.

PubMed

De Zoysa, Meemanage D; Wu, Guowei; Katz, Raviv; Yu, Yi-Tao

2018-06-05

Box H/ACA RNAs are a group of small RNAs found in abundance in eukaryotes (as well as in archaea). Although their sequences differ, eukaryotic box H/ACA RNAs all share the same unique hairpin-hinge-hairpin-tail structure. Almost all of them function as guides that primarily direct pseudouridylation of rRNAs and spliceosomal snRNAs at specific sites. Although box H/ACA RNA-guided pseudouridylation has been extensively studied, the detailed rules governing this reaction, especially those concerning the guide RNA-substrate RNA base-pairing interactions that determine the specificity and efficiency of pseudouridylation, are still not exactly clear. This is particularly relevant given that the lengths of the guide sequences involved in base-pairing vary from one box H/ACA RNA to another. Here, we carry out a detailed investigation into guide-substrate base-pairing interactions, and identify the minimum number of base-pairs (8), required for RNA-guided pseudouridylation. In addition, we find that the pseudouridylation pocket, present in each hairpin of box H/ACA RNA, exhibits flexibility in fitting slightly different substrate sequences. Our results are consistent across three independent pseudouridylation pockets tested, suggesting that our findings are generally applicable to box H/ACA RNA-guided RNA pseudouridylation. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

PubMed

Xi, Qian-Yun; Xiong, Yuan-Yan; Wang, Yuan-Mei; Cheng, Xiao; Qi, Qi-En; Shu, Gang; Wang, Song-Bo; Wang, Li-Na; Gao, Ping; Zhu, Xiao-Tong; Jiang, Qing-Yan; Zhang, Yong-Liang; Liu, Li

2015-01-01

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Quantitative analyses and transcriptomic profiling of circulating messenger RNAs as biomarkers of rat liver injury.

PubMed

Wetmore, Barbara A; Brees, Dominique J; Singh, Reetu; Watkins, Paul B; Andersen, Melvin E; Loy, James; Thomas, Russell S

2010-06-01

Serum aminotransferases have been the clinical standard for evaluating liver injury for the past 50-60 years. These tissue enzymes lack specificity, also tracking injury to other tissues. New technologies assessing tissue-specific messenger RNA (mRNA) release into blood should provide greater specificity and permit indirect assessment of gene expression status of injured tissue. To evaluate the potential of circulating mRNAs as biomarkers of liver injury, rats were treated either with hepatotoxic doses of D-(+)-galactosamine (DGAL) or acetaminophen (APAP) or a myotoxic dose of bupivacaine HCl (BPVC). Plasma, serum, and liver samples were obtained from each rat. Serum alanine aminotransferase and aspartate aminotransferase were increased by all three compounds, whereas circulating liver-specific mRNAs were only increased by the hepatotoxicants. With APAP, liver-specific mRNAs were significantly increased in plasma at doses that had no effect on serum aminotransferases or liver histopathology. Characterization of the circulating mRNAs by sucrose density gradient centrifugation revealed that the liver-specific mRNAs were associated with both necrotic debris and microvesicles. DGAL treatment also induced a shift in the size of plasma microvesicles, consistent with active release of microvesicles following liver injury. Finally, gene expression microarray analysis of the plasma following DGAL and APAP treatment revealed chemical-specific profiles. The comparative analysis of circulating liver mRNAs with traditional serum transaminases and histopathology indicated that the circulating liver mRNAs were more specific and more sensitive biomarkers of liver injury. Further, the possibility of identifying chemical-specific transcriptional profiles from circulating mRNAs could open a range of possibilities for identifying the etiology of drug/chemical-induced liver injury.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening.

PubMed

Mensah, Mavis; Borzi, Cristina; Verri, Carla; Suatoni, Paola; Conte, Davide; Pastorino, Ugo; Orazio, Fortunato; Sozzi, Gabriella; Boeri, Mattia

2017-10-26

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

Circular RNA: a new star in neurological diseases.

PubMed

Li, Tao-Ran; Jia, Yan-Jie; Wang, Qun; Shao, Xiao-Qiu; Lv, Rui-Juan

2017-08-01

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs characterized by the presence of a covalent bond linking the 3' and 5' ends generated by backsplicing. In this review, we summarize a number of the latest theories regarding the biogenesis, properties and functions of circRNAs. Specifically, we focus on the advancing characteristics and functions of circRNAs in the brain and neurological diseases. CircRNAs exhibit the characteristics of species conservation, abundance and tissue/developmental-stage-specific expression in the brain. We also describe the relationship between circRNAs and several neurological diseases and highlight their functions in neurological diseases.

Identification and profiling of Cyprinus carpio microRNAs during ovary differentiation by deep sequencing.

PubMed

Wang, Fang; Jia, Yongfang; Wang, Po; Yang, Qianwen; Du, QiYan; Chang, ZhongJie

2017-04-28

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression by targeting specific mRNAs. However, the possible role of miRNAs in the ovary differentiation and development of fish is not well understood. In this study, we examined the expression profiles and differential expression of miRNAs during three key stages of ovarian development and different developmental stages in common carp Cyprinus carpio. A total of 8765 miRNAs were identified, including 2155 conserved miRNAs highly conserved among various species, 145 miRNAs registered in miRBase for common carp, and 6505 novel miRNAs identified in common carp for the first time. Comparison of miRNA expression profiles among the five libraries identified 714 co-expressed and 2382 specific expressed miRNAs. Overall, 150, 628, and 431 specifically expressed miRNAs were identified in primordial gonad, juvenile ovary, and adult ovary, respectively. MiR-6758-3p, miR-3050-5p, and miR-2985-3p were highly expressed in primordial gonad, miR-3544-5p, miR-6877-3p, and miR-9086-5p were highly expressed in juvenile ovary, and miR-154-3p, miR-5307-5p, and miR-3958-3p were highly expressed in adult ovary. Predicted target genes of specific miRNAs in primordial gonad were involved in many reproductive biology signaling pathways, including transforming growth factor-β, Wnt, oocyte meiosis, mitogen-activated protein kinase, Notch, p53, and gonadotropin-releasing hormone pathways. Target-gene prediction revealed upward trends in miRNAs targeting male-bias genes, including dmrt1, atm, gsdf, and sox9, and downward trends in miRNAs targeting female-bias genes including foxl2, smad3, and smad4. Other sex-related genes such as sf1 were also predicted to be miRNA target genes. This comprehensive miRNA transcriptome analysis demonstrated differential expression profiles of miRNAs during ovary development in common carp. These results could facilitate future exploitation of the sex-regulatory roles and mechanisms of miRNAs, especially in primordial gonads, while the specifically expressed miRNAs represent candidates for studying the mechanisms of ovary determination in Yellow River carp.

MicroRNAs: A novel potential biomarker for diagnosis and therapy in patients with non-small cell lung cancer.

PubMed

Zhou, Qun; Huang, Shao-Xin; Zhang, Feng; Li, Shu-Jun; Liu, Cong; Xi, Yong-Yong; Wang, Liang; Wang, Xin; He, Qi-Qiang; Sun, Cheng-Cao; Li, De-Jia

2017-12-01

Lung cancer is still one of the most serious causes of cancer-related deaths all over the world. MicroRNAs (miRNAs) are defined as small non-coding RNAs which could play a pivotal role in post-transcriptional regulation of gene expression. Increasing evidence demonstrated dysregulation of miRNA expression associates with the development and progression of NSCLC. To emphasize a variety of tissue-specific miRNAs, circulating miRNAs and miRNA-derived exosomes could be used as potential diagnostic and therapeutic biomarkers in NSCLC patients. In the current review, we paid attention to the significant discoveries of preclinical and clinical studies, which performed on tissue-specific miRNA, circulating miRNA and exosomal miRNA. The related studies were obtained through a systematic search of Pubmed, Web of Science, Embase. A variety of tissue-specific miRNAs and circulating miRNAs with high sensitivity and specificity which could be used as potential diagnostic and therapeutic biomarkers in NSCLC patients. In addition, we emphasize that the miRNA-derived exosomes become novel diagnostic biomarkers potentially in these patients with NSCLC. MiRNAs have emerged as non-coding RNAs, which have potential to be candidates for the diagnosis and therapy of NSCLC. © 2017 John Wiley & Sons Ltd.

MicroRNAs Change the Landscape of Cancer Resistance.

PubMed

Zhu, Jun; Zhu, Wei; Wu, Wei

2018-01-01

One of the major challenges in the cancer treatment is the development of drug resistance. It represents a major obstacle to curing cancer with constrained efficacy of both conventional chemotherapy and targeted therapies, even recent immune checkpoint blockade therapy. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in the resistance to various cancer treatments. MicroRNAs are a family of small noncoding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified in human genome. While one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance, thereby modulating the sensitivity of cancer cells to treatment. Therefore, manipulation of miRNAs may be an attractive strategy for more effective individualized therapies through reprograming resistant network in cancer cells.

Solexa Sequencing of Novel and Differentially Expressed MicroRNAs in Testicular and Ovarian Tissues in Holstein Cattle

PubMed Central

Huang, Jinming; Ju, Zhihua; Li, Qiuling; Hou, Qinlei; Wang, Changfa; Li, Jianbin; Li, Rongling; Wang, Lingling; Sun, Tao; Hang, Suqin; Gao, Yundong; Hou, Minghai; Zhong, Jifeng

2011-01-01

The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle. PMID:21912509

Lung Cancer-Specific Circular RNAs as Biomarkers

DTIC Science & Technology

2017-08-01

Award Number: W81XWH-16-1-0239 TITLE: Lung Cancer-Specific Circular RNAs as Biomarkers PRINCIPAL INVESTIGATOR: Yin-Yuan Mo CONTRACTING...Specific Circular RNAs as Biomarkers 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-16-1-0239 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Yin-Yuan Mo Betty...14. ABSTRACT The major goal of this application is to determine whether lung cancer cells differentially express circular RNAs such that these

Transposable elements (TEs) contribute to stress-related long intergenic noncoding RNAs in plants.

PubMed

Wang, Dong; Qu, Zhipeng; Yang, Lan; Zhang, Qingzhu; Liu, Zhi-Hong; Do, Trung; Adelson, David L; Wang, Zhen-Yu; Searle, Iain; Zhu, Jian-Kang

2017-04-01

Noncoding RNAs have been extensively described in plant and animal transcriptomes by using high-throughput sequencing technology. Of these noncoding RNAs, a growing number of long intergenic noncoding RNAs (lincRNAs) have been described in multicellular organisms, however the origins and functions of many lincRNAs remain to be explored. In many eukaryotic genomes, transposable elements (TEs) are widely distributed and often account for large fractions of plant and animal genomes yet the contribution of TEs to lincRNAs is largely unknown. By using strand-specific RNA-sequencing, we profiled the expression patterns of lincRNAs in Arabidopsis, rice and maize, and identified 47 611 and 398 TE-associated lincRNAs (TE-lincRNAs), respectively. TE-lincRNAs were more often derived from retrotransposons than DNA transposons and as retrotransposon copy number in both rice and maize genomes so did TE-lincRNAs. We validated the expression of these TE-lincRNAs by strand-specific RT-PCR and also demonstrated tissue-specific transcription and stress-induced TE-lincRNAs either after salt, abscisic acid (ABA) or cold treatments. For Arabidopsis TE-lincRNA11195, mutants had reduced sensitivity to ABA as demonstrated by longer roots and higher shoot biomass when compared to wild-type. Finally, by altering the chromatin state in the Arabidopsis chromatin remodelling mutant ddm1, unique lincRNAs including TE-lincRNAs were generated from the preceding untranscribed regions and interestingly inherited in a wild-type background in subsequent generations. Our findings not only demonstrate that TE-associated lincRNAs play important roles in plant abiotic stress responses but lincRNAs and TE-lincRNAs might act as an adaptive reservoir in eukaryotes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

PubMed

Romanienko, Peter J; Giacalone, Joseph; Ingenito, Joanne; Wang, Yijie; Isaka, Mayumi; Johnson, Thomas; You, Yun; Mark, Willie H

2016-01-01

The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

[Efficacy of siRNA on feline leukemia virus replication in vitro].

PubMed

Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

2015-01-01

Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments.

PubMed

Zhou, Bailing; Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu; Yang, Yuedong; Zhou, Yaoqi; Wang, Jihua

2018-01-04

Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments

PubMed Central

Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu

2018-01-01

Abstract Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. PMID:28985416

RNA Export through the NPC in Eukaryotes.

PubMed

Okamura, Masumi; Inose, Haruko; Masuda, Seiji

2015-03-20

In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

RNA Export through the NPC in Eukaryotes

PubMed Central

Okamura, Masumi; Inose, Haruko; Masuda, Seiji

2015-01-01

In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC. PMID:25802992

MicroRNAs derived from circulating exosomes as non-invasive biomarkers for screening and diagnose lung cancer

PubMed Central

Cazzoli, Riccardo; Buttitta, Fiamma; Di Nicola, Marta; Malatesta, Sara; Marchetti, Antonio; Pass, Harvey I.

2013-01-01

Introduction Lung cancer is formerly the highest cause of mortality among tumor pathologies worldwide. There are no validated techniques for an early detection of pulmonary cancer lesions other than low-dose helical CT-scan. Unfortunately, this method have some downside effects. Recent studies have laid the basis for development of exosomes-based techniques to screen/diagnose lung cancers. As the isolation of circulating exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostic applications. Methods We used a first set of 30 plasma samples from as many patients, including 10 patients affected by Lung Adenocarcinomas, 10 with Lung Granulomas and 10 healthy smokers matched for age and sex as negative controls. Wide range microRNAs analysis (742 microRNAs) was performed by quantitative RT-PCR. Data were compared by lesion characteristics using WEKA software for statistics and modeling. Subsequently, selected microRNAs were evaluated on an independent larger group of samples (105 specimens: 50 Lung Adenocarcinomas, 30 Lung Granulomas and 25 healthy smokers). Results This analysis led to the selection of 4 microRNAs to perform a screening test (miR-378a, miR-379, miR-139-5p and miR-200b-5p), useful to divide population into 2 groups: nodule (lung adenocarcinomas+carcinomas) and non-nodule (healthy former smokers). Six microRNAs (miR-151a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100 and miR-154-3p) were selected for a second test on the “nodule” population to discriminate between lung adenocarcinoma and granuloma. Conclusions “Screening test” has shown 97.5% sensitivity, 72.0% specificity, AUC ROC of 90.8%. “Diagnostic test” had 96.0% sensitivity, 60.0% specificity, AUC ROC of 76.0%. Further evaluation is needed to confirm the predictive power of those models on higher cohorts of samples. PMID:23945385

Gene-Specific Demethylation as Targeted Therapy in MDS

DTIC Science & Technology

2016-07-01

methylation remain elusive. This proposal builds on our recent discovery of a novel class of RNAs , the DiRs or DNMT1-interacting RNAs , involved in...cell type-specific DNA methylation patterns. Based on these findings, we hypothesize that DNA methylation changes can be corrected by RNAs . We aim to...aberrant DNA methylation remain elusive. This proposal builds on our recent discovery of a novel class of RNAs , the DiRs or DNMT1-interacting RNAs

Circulating Plasma and Exosomal microRNAs as Indicators of Drug-Induced Organ Injury in Rodent Models

PubMed Central

Cho, Young-Eun; Kim, Sang-Hyun; Lee, Byung-Heon; Baek, Moon-Chang

2017-01-01

This study was performed to evaluate whether microRNAs (miRNAs) in circulating exosomes may serve as biomarkers of drug-induced liver, kidney, or muscle-injury. Quantitative PCR analyses were performed to measure the amounts of liver-specific miRNAs (miR-122, miR-192, and miR-155), kidney-specific miR-146a, or muscle-specific miR-206 in plasma and exosomes from mice treated with liver, kidney or muscle toxicants. The levels of liver-specific miRNAs in circulating plasma and exosomes were elevated in acetaminophen-induced liver injury and returned to basal levels by treatment with antioxidant N-acetyl-cysteine. Circulating miR-146a and miR-206 were increased in cisplatin-induced nephrotoxicity and bupivacaine-induced myotoxicity, respectively. Taken together, these results indicate that circulating plasma and exosomal miRNAs can be used as potential biomarkers specific for drug-induced liver, kidney or muscle injury. PMID:28208010

Profiling and Validation of the Circular RNA Repertoire in Adult Murine Hearts.

PubMed

Jakobi, Tobias; Czaja-Hasse, Lisa F; Reinhardt, Richard; Dieterich, Christoph

2016-08-01

For several decades, cardiovascular disease has been the leading cause of death throughout all countries. There is a strong genetic component to many disease subtypes (e.g., cardiomyopathy) and we are just beginning to understand the relevant genetic factors. Several studies have related RNA splicing to cardiovascular disease and circular RNAs (circRNAs) are an emerging player. circRNAs, which originate through back-splicing events from primary transcripts, are resistant to exonucleases and typically not polyadenylated. Initial functional studies show clear phenotypic outcomes for selected circRNAs. We provide, for the first time, a comprehensive catalogue of RNase R-resistant circRNA species for the adult murine heart. This work combines state-of-the-art circle sequencing with our novel DCC software to explore the circRNA landscape of heart tissue. Overall, we identified 575 circRNA species that pass a beta-binomial test for enrichment (false discovery rate of 1%) in the exonuclease-treated sequencing sample. Several circRNAs can be directly attributed to host genes that have been previously described as associated with cardiovascular disease. Further studies of these candidate circRNAs may reveal disease-relevant properties or functions of specific circRNAs. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Long noncoding RNA in hematopoiesis and immunity.

PubMed

Satpathy, Ansuman T; Chang, Howard Y

2015-05-19

Dynamic gene expression during cellular differentiation is tightly coordinated by transcriptional and post-transcriptional mechanisms. An emerging theme is the central role of long noncoding RNAs (lncRNAs) in the regulation of this specificity. Recent advances demonstrate that lncRNAs are expressed in a lineage-specific manner and control the development of several cell types in the hematopoietic system. Moreover, specific lncRNAs are induced to modulate innate and adaptive immune responses. lncRNAs can function via RNA-DNA, RNA-RNA, and RNA-protein target interactions. As a result, they affect several stages of gene regulation, including chromatin modification, mRNA biogenesis, and protein signaling. We discuss recent advances, future prospects, and challenges in understanding the roles of lncRNAs in immunity and immune-mediated diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Deep experimental profiling of microRNA diversity, deployment, and evolution across the Drosophila genus.

PubMed

Mohammed, Jaaved; Flynt, Alex S; Panzarino, Alexandra M; Mondal, Md Mosharrof Hossein; DeCruz, Matthew; Siepel, Adam; Lai, Eric C

2018-01-01

To assess miRNA evolution across the Drosophila genus, we analyzed several billion small RNA reads across 12 fruit fly species. These data permit comprehensive curation of species- and clade-specific variation in miRNA identity, abundance, and processing. Among well-conserved miRNAs, we observed unexpected cases of clade-specific variation in 5' end precision, occasional antisense loci, and putatively noncanonical loci. We also used strict criteria to identify a large set (649) of novel, evolutionarily restricted miRNAs. Within the bulk collection of species-restricted miRNAs, two notable subpopulations are splicing-derived mirtrons and testes-restricted, recently evolved, clustered (TRC) canonical miRNAs. We quantified miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover. We observed striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identified flux heterogeneity among Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. Mirtrons are associated with high rates of 3' untemplated addition, a mechanism that impedes their biogenesis, whereas TRC miRNAs appear to evolve under positive selection. Altogether, these data reveal miRNA diversity among Drosophila species and principles underlying their emergence and evolution. © 2018 Mohammed et al.; Published by Cold Spring Harbor Laboratory Press.

Global Analysis of Small RNA Dynamics during Seed Development of Picea glauca and Arabidopsis thaliana Populations Reveals Insights on their Evolutionary Trajectories

PubMed Central

Liu, Yang; El-Kassaby, Yousry A.

2017-01-01

While DNA methylation carries genetic signals and is instrumental in the evolution of organismal complexity, small RNAs (sRNAs), ~18–24 ribonucleotide (nt) sequences, are crucial mediators of methylation as well as gene silencing. However, scant study deals with sRNA evolution via featuring their expression dynamics coupled with species of different evolutionary time. Here we report an atlas of sRNAs and microRNAs (miRNAs, single-stranded sRNAs) produced over time at seed-set of two major spermatophytes represented by populations of Picea glauca and Arabidopsis thaliana with different seed-set duration. We applied diverse profiling methods to examine sRNA and miRNA features, including size distribution, sequence conservation and reproduction-specific regulation, as well as to predict their putative targets. The top 27 most abundant miRNAs were highly overlapped between the two species (e.g., miR166,−319 and−396), but in P. glauca, they were less abundant and significantly less correlated with seed-set phases. The most abundant sRNAs in libraries were deeply conserved miRNAs in the plant kingdom for Arabidopsis but long sRNAs (24-nt) for P. glauca. We also found significant difference in normalized expression between populations for population-specific sRNAs but not for lineage-specific ones. Moreover, lineage-specific sRNAs were enriched in the 21-nt size class. This pattern is consistent in both species and alludes to a specific type of sRNAs (e.g., miRNA, tasiRNA) being selected for. In addition, we deemed 24 and 9 sRNAs in P. glauca and Arabidopsis, respectively, as sRNA candidates targeting known adaptive genes. Temperature had significant influence on selected gene and miRNA expression at seed development in both species. This study increases our integrated understanding of sRNA evolution and its potential link to genomic architecture (e.g., sRNA derivation from genome and sRNA-mediated genomic events) and organismal complexity (e.g., association between different sRNA expression and their functionality). PMID:29046688

A novel microRNA assay with optical detection and enzyme-free DNA circuits

NASA Astrophysics Data System (ADS)

Liao, Yuhui; Zhou, Xiaoming

2014-09-01

MicroRNAs (miRNAs) participate in the significant processes of life course, can be used as quantificational biomarkers for cellular level researches and related diseases. Conventional methods of miRNAs' quantitative detection are obsessed with low sensitivity, time and labour consuming. Otherwise, the emerging miRNAs detection approaches are mostly exposed to the expensive equipment demands and the professional operation, remains at the stage of laboratory and concept demonstration phase. Herein, we designed a novel miRNAs detection platform that based on enzyme-free DNA circuits and electrochemical luminescence (ECL). MicroRNA21 was chosen as the ideal analyte of this platform. The whole process consists of enzyme-free DNA circuits and ECL signal giving-out steps, achieves advantages of operating in constant temperature condition, without the participation of the enzyme, preferable sensitivity and specificity. Through this approach, the sensitivity achieved at 10pM. It is indicated that this miRNAs detection platform possesses potentials to be an innovation of miRNA detection technologies in routine tests. Benefits of the high penetration of ECL in well-equipped medical establishment, this approach could greatly lessen the obstacles in process of popularization and possess excellent prospects of practical application.

Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone

PubMed Central

Melia, Tisha; Hao, Pengying; Yilmaz, Feyza

2015-01-01

Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the expression of sex-specific lincRNAs and their potential for regulation of sex differences in liver physiology and disease. PMID:26459762

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

PubMed Central

Rousseau, Julie; Gioia, Roberta; Layrolle, Pierre; Lieubeau, Blandine; Heymann, Dominique; Rossi, Antonio; Marini, Joan C; Trichet, Valerie; Forlino, Antonella

2014-01-01

Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation. PMID:24022296

Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood

PubMed Central

Peters, Oliver; Rajewsky, Nikolaus

2015-01-01

Covalently closed circular RNA molecules (circRNAs) have recently emerged as a class of RNA isoforms with widespread and tissue specific expression across animals, oftentimes independent of the corresponding linear mRNAs. circRNAs are remarkably stable and sometimes highly expressed molecules. Here, we sequenced RNA in human peripheral whole blood to determine the potential of circRNAs as biomarkers in an easily accessible body fluid. We report the reproducible detection of thousands of circRNAs. Importantly, we observed that hundreds of circRNAs are much higher expressed than corresponding linear mRNAs. Thus, circRNA expression in human blood reveals and quantifies the activity of hundreds of coding genes not accessible by classical mRNA specific assays. Our findings suggest that circRNAs could be used as biomarker molecules in standard clinical blood samples. PMID:26485708

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

PubMed

Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

PubMed

Nguyen, Thao T; Brenu, Ekua W; Staines, Don R; Marshall-Gradisnik, Sonya M

2014-01-01

MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases.

MicroRNA-122 regulates polyploidization in the murine liver.

PubMed

Hsu, Shu-Hao; Delgado, Evan R; Otero, P Anthony; Teng, Kun-Yu; Kutay, Huban; Meehan, Kolin M; Moroney, Justin B; Monga, Jappmann K; Hand, Nicholas J; Friedman, Joshua R; Ghoshal, Kalpana; Duncan, Andrew W

2016-08-01

A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ∼90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy, we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Second, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, lifelong depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated overexpression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l, and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified; our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization, and these studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis, and disease. (Hepatology 2016;64:599-615). © 2016 by the American Association for the Study of Liver Diseases.

MicroRNA-122 Regulates Polyploidization in the Murine Liver

PubMed Central

Hsu, Shu-hao; Delgado, Evan R.; Otero, P. Anthony; Teng, Kun-yu; Kutay, Huban; Meehan, Kolin M.; Moroney, Justin B.; Monga, Jappmann K.; Hand, Nicholas J.; Friedman, Joshua R.; Ghoshal, Kalpana; Duncan, Andrew W.

2016-01-01

A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2–3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. PMID:27016325

Dissecting microRNA dysregulation in age-related macular degeneration: new targets for eye gene therapy.

PubMed

Askou, Anne Louise; Alsing, Sidsel; Holmgaard, Andreas; Bek, Toke; Corydon, Thomas J

2018-02-01

MicroRNAs (miRNAs) are key regulators of gene expression in humans. Overexpression or depletion of individual miRNAs is associated with human disease. Current knowledge suggests that the retina is influenced by miRNAs and that dysregulation of miRNAs as well as alterations in components of the miRNA biogenesis machinery are involved in retinal diseases, including age-related macular degeneration (AMD). Furthermore, recent studies have indicated that the vitreous has a specific panel of circulating miRNAs and that this panel varies according to the specific pathological stress experienced by the retinal cells. MicroRNA (miRNA) profiling indicates subtype-specific miRNA profiles for late-stage AMD highlighting the importance of proper miRNA regulation in AMD. This review will describe the function of important miRNAs involved in inflammation, oxidative stress and pathological neovascularization, the key molecular mechanisms leading to AMD, and focus on dysregulated miRNAs as potential therapeutic targets in AMD. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

MicroRNAs and their roles in aging

PubMed Central

Smith-Vikos, Thalyana; Slack, Frank J.

2012-01-01

MicroRNAs (miRNAs) are a class of short non-coding RNAs that bind mRNAs through partial base-pair complementarity with their target genes, resulting in post-transcriptional repression of gene expression. The role of miRNAs in controlling aging processes has been uncovered recently with the discovery of miRNAs that regulate lifespan in the nematode Caenorhabditis elegans through insulin and insulin-like growth factor-1 signaling and DNA damage checkpoint factors. Furthermore, numerous miRNAs are differentially expressed during aging in C. elegans, but the specific functions of many of these miRNAs are still unknown. Recently, various miRNAs have been identified that are up- or down-regulated during mammalian aging by comparing their tissue-specific expression in younger and older mice. In addition, many miRNAs have been implicated in governing senescence in a variety of human cell lines, and the precise functions of some of these miRNAs in regulating cellular senescence have helped to elucidate mechanisms underlying aging. In this Commentary, we review the various regulatory roles of miRNAs during aging processes. We highlight how certain miRNAs can regulate aging on the level of organism lifespan, tissue aging or cellular senescence. Finally, we discuss future approaches that might be used to investigate the mechanisms by which miRNAs govern aging processes. PMID:22294612

Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus

PubMed Central

2011-01-01

Background MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. Results To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. Conclusions Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs. PMID:21699734

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

Long Non-Coding RNAs in Haematological Malignancies

PubMed Central

Garitano-Trojaola, Andoni; Agirre, Xabier; Prósper, Felipe; Fortes, Puri

2013-01-01

Long non-coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. LncRNAs are as diverse as mRNAs and they normally share the same biosynthetic machinery based on RNA polymerase II, splicing and polyadenylation. However, lncRNAs have low coding potential. Compared to mRNAs, lncRNAs are preferentially nuclear, more tissue specific and expressed at lower levels. Most of the lncRNAs described to date modulate the expression of specific genes by guiding chromatin remodelling factors; inducing chromosomal loopings; affecting transcription, splicing, translation or mRNA stability; or serving as scaffolds for the organization of cellular structures. They can function in cis, cotranscriptionally, or in trans, acting as decoys, scaffolds or guides. These functions seem essential to allow cell differentiation and growth. In fact, many lncRNAs have been shown to exert oncogenic or tumor suppressor properties in several cancers including haematological malignancies. In this review, we summarize what is known about lncRNAs, the mechanisms for their regulation in cancer and their role in leukemogenesis, lymphomagenesis and hematopoiesis. Furthermore, we discuss the potential of lncRNAs in diagnosis, prognosis and therapy in cancer, with special attention to haematological malignancies. PMID:23887658

Screening of lymph nodes metastasis associated lncRNAs in colorectal cancer patients

PubMed Central

Han, Jun; Rong, Long-Fei; Shi, Chuan-Bin; Dong, Xiao-Gang; Wang, Jie; Wang, Bao-Lin; Wen, Hao; He, Zhen-Yu

2014-01-01

AIM: To screen lymph nodes metastasis associated long noncoding RNAs (lncRNAs) in colorectal cancer through microarray analysis. METHODS: Metastatic lymph node (MLN), normal lymph node (NLN) and tumor tissues of 3 colorectal cancer (CRC) patients were collected during the operation and validated by pathological examinations. RNAs were extracted from MLN, NLN, and cancer tissues separately. RNA quantity and quality were measured with a NanoDrop ND-1000 spectrophotometer and RNA integrity was assessed by standard denaturing agarose electrophoresis. Agilent Feature Extraction Software (Version 11.0.1.1) was used to analyze acquired array images. Four differently expressed lncRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) in 26 subsets of MLN, NLN, and tumor tissues. RESULTS: Of 33045 lncRNAs, 1133 were differentially expressed in MLN compared with NLN, of which 260 were up-regulated and 873 down-regulated (≥ 2 fold-change). Five hundred and forty-five lncRNAs were differentially expressed in MLN compared with tumor tissues, of which 460 were up-regulated and 85 down-regulated (≥ 2 fold-change). Compared with NLN and cancer tissues, 14 lncRNAs were specifically up-regulated and 5 specifically down-regulated in MLN. AK307796, ENST00000425785, and AK021444 were confirmed to be specifically up-regulated in MLN and ENST00000465846 specifically down-regulated in MLN by qRT-PCR in 26 CRC patients. CONCLUSION: The specifically expressed lncRNAs in MLN may exert a partial or key role in the progress of lymph nodes metastasis of CRC. PMID:25009386

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs*

PubMed Central

Ortogero, Nicole; Schuster, Andrew S.; Oliver, Daniel K.; Riordan, Connor R.; Hong, Annie S.; Hennig, Grant W.; Luong, Dickson; Bao, Jianqiang; Bhetwal, Bhupal P.; Ro, Seungil; McCarrey, John R.; Yan, Wei

2014-01-01

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs. PMID:25320077

Functional analysis of neuronal microRNAs in Caenorhabditis elegans dauer formation by combinational genetics and Neuronal miRISC immunoprecipitation.

PubMed

Than, Minh T; Kudlow, Brian A; Han, Min

2013-06-01

Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic "enhancer" approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

snoSeeker: an advanced computational package for screening of guide and orphan snoRNA genes in the human genome.

PubMed

Yang, Jian-Hua; Zhang, Xiao-Chen; Huang, Zhan-Peng; Zhou, Hui; Huang, Mian-Bo; Zhang, Shu; Chen, Yue-Qin; Qu, Liang-Hu

2006-01-01

Small nucleolar RNAs (snoRNAs) represent an abundant group of non-coding RNAs in eukaryotes. They can be divided into guide and orphan snoRNAs according to the presence or absence of antisense sequence to rRNAs or snRNAs. Current snoRNA-searching programs, which are essentially based on sequence complementarity to rRNAs or snRNAs, exist only for the screening of guide snoRNAs. In this study, we have developed an advanced computational package, snoSeeker, which includes CDseeker and ACAseeker programs, for the highly efficient and specific screening of both guide and orphan snoRNA genes in mammalian genomes. By using these programs, we have systematically scanned four human-mammal whole-genome alignment (WGA) sequences and identified 54 novel candidates including 26 orphan candidates as well as 266 known snoRNA genes. Eighteen novel snoRNAs were further experimentally confirmed with four snoRNAs exhibiting a tissue-specific or restricted expression pattern. The results of this study provide the most comprehensive listing of two families of snoRNA genes in the human genome till date.

Ensemble-based classification approach for micro-RNA mining applied on diverse metagenomic sequences.

PubMed

ElGokhy, Sherin M; ElHefnawi, Mahmoud; Shoukry, Amin

2014-05-06

MicroRNAs (miRNAs) are endogenous ∼22 nt RNAs that are identified in many species as powerful regulators of gene expressions. Experimental identification of miRNAs is still slow since miRNAs are difficult to isolate by cloning due to their low expression, low stability, tissue specificity and the high cost of the cloning procedure. Thus, computational identification of miRNAs from genomic sequences provide a valuable complement to cloning. Different approaches for identification of miRNAs have been proposed based on homology, thermodynamic parameters, and cross-species comparisons. The present paper focuses on the integration of miRNA classifiers in a meta-classifier and the identification of miRNAs from metagenomic sequences collected from different environments. An ensemble of classifiers is proposed for miRNA hairpin prediction based on four well-known classifiers (Triplet SVM, Mipred, Virgo and EumiR), with non-identical features, and which have been trained on different data. Their decisions are combined using a single hidden layer neural network to increase the accuracy of the predictions. Our ensemble classifier achieved 89.3% accuracy, 82.2% f-measure, 74% sensitivity, 97% specificity, 92.5% precision and 88.2% negative predictive value when tested on real miRNA and pseudo sequence data. The area under the receiver operating characteristic curve of our classifier is 0.9 which represents a high performance index.The proposed classifier yields a significant performance improvement relative to Triplet-SVM, Virgo and EumiR and a minor refinement over MiPred.The developed ensemble classifier is used for miRNA prediction in mine drainage, groundwater and marine metagenomic sequences downloaded from the NCBI sequence reed archive. By consulting the miRBase repository, 179 miRNAs have been identified as highly probable miRNAs. Our new approach could thus be used for mining metagenomic sequences and finding new and homologous miRNAs. The paper investigates a computational tool for miRNA prediction in genomic or metagenomic data. It has been applied on three metagenomic samples from different environments (mine drainage, groundwater and marine metagenomic sequences). The prediction results provide a set of extremely potential miRNA hairpins for cloning prediction methods. Among the ensemble prediction obtained results there are pre-miRNA candidates that have been validated using miRbase while they have not been recognized by some of the base classifiers.

Expression profile of circular RNAs in infantile hemangioma detected by RNA-Seq.

PubMed

Li, Jun; Li, Qian; Chen, Ling; Gao, Yanli; Li, Jingyun

2018-05-01

Circular RNAs (circRNAs) have emerged as a novel class of widespread non-coding RNAs, and they play crucial roles in various biological processes. However, the characterization and function of circRNAs in infantile hemangioma (IH) remain elusive. In this study, we used RNA-Seq and circRNA prediction to study and characterize the circRNAs in IH tissue and a matched normal skin control. Specific circRNAs were verified using real-time polymerase chain reaction. We found that of the 9811 identified circRNAs, 249 candidates were differentially expressed, including 124 upregulated and 125 downregulated circRNAs in the IH group compared with the matched normal skin control group. A set of differentially expressed circRNAs (in particular, hsa_circRNA001885 and hsa_circRNA006612 expression) were confirmed using qRT-PCR. Gene ontology and pathway analysis revealed that compared to matched normal skin tissues, many processes that were over-represented in IH group were related to the binding, protein binding, gap junction, and focal adhesion. Specific circRNAs were associated with several micro-RNAs (miRNAs) predicted using miRanda. Altogether, our findings highlight the potential importance of circRNAs in the biology of IH and its response to treatment.

Song exposure regulates known and novel microRNAs in the zebra finch auditory forebrain

PubMed Central

2011-01-01

Background In an important model for neuroscience, songbirds learn to discriminate songs they hear during tape-recorded playbacks, as demonstrated by song-specific habituation of both behavioral and neurogenomic responses in the auditory forebrain. We hypothesized that microRNAs (miRNAs or miRs) may participate in the changing pattern of gene expression induced by song exposure. To test this, we used massively parallel Illumina sequencing to analyse small RNAs from auditory forebrain of adult zebra finches exposed to tape-recorded birdsong or silence. Results In the auditory forebrain, we identified 121 known miRNAs conserved in other vertebrates. We also identified 34 novel miRNAs that do not align to human or chicken genomes. Five conserved miRNAs showed significant and consistent changes in copy number after song exposure across three biological replications of the song-silence comparison, with two increasing (tgu-miR-25, tgu-miR-192) and three decreasing (tgu-miR-92, tgu-miR-124, tgu-miR-129-5p). We also detected a locus on the Z sex chromosome that produces three different novel miRNAs, with supporting evidence from Northern blot and TaqMan qPCR assays for differential expression in males and females and in response to song playbacks. One of these, tgu-miR-2954-3p, is predicted (by TargetScan) to regulate eight song-responsive mRNAs that all have functions in cellular proliferation and neuronal differentiation. Conclusions The experience of hearing another bird singing alters the profile of miRNAs in the auditory forebrain of zebra finches. The response involves both known conserved miRNAs and novel miRNAs described so far only in the zebra finch, including a novel sex-linked, song-responsive miRNA. These results indicate that miRNAs are likely to contribute to the unique behavioural biology of learned song communication in songbirds. PMID:21627805

TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

PubMed

Bandyopadhyay, Sanghamitra; Mitra, Ramkrishna

2009-10-15

Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the same. Again, when an existing method (NBmiRTar) is executed with the our proposed negative data, we observe an improvement in its performance. These clearly establish the effectiveness of the proposed approach of selecting the negative examples systematically. TargetMiner is now available as an online tool at www.isical.ac.in/ approximately bioinfo_miu

Small RNA sorting: matchmaking for Argonautes

PubMed Central

Czech, Benjamin; Hannon, Gregory J.

2013-01-01

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. PMID:21116305

Identification of Prostate Cancer-Specific Circular RNAs

DTIC Science & Technology

2016-12-01

they often have regulatory functions. For example, circRNAs can serve as endogenous microRNA sponges to neutralize the microRNA function. However, it...circRNAs is the capability to serve as sponges to neutralize the endogenous microRNAs. In this regard, all of these circRNAs had the potential to

Identification of Prostate Cancer-Specific Circular RNAs

DTIC Science & Technology

2015-10-01

AND SUBTITLE Identification of Prostate Cancer-Specific Circular RNAs ” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0384 5c. PROGRAM ELEMENT...Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The major goal of this application is to determine whether newly identified circular RNAs can

MiRNAs in bone diseases.

PubMed

Moore, Benjamin T; Xiao, Peng

2013-01-01

MicroRNAs (miRNAs), which mainly inhibit protein expression by targeting the 3'UTR (untranslated region) of mRNAs, are known to play various roles in the pathogenesis of many different types of diseases. Specifically, in bone diseases, recent emphasis has been placed on the involvement of miRNAs in the differentiation and proliferation of bone and cartilage cells, particularly with regards to how these mechanisms contribute to bone homeostasis. In this review, we summarize miRNAs that are important in the differentiation and proliferation of bone cells, and specific miRNAs associated with bone diseases, such as osteoporosis, osteoarthritis and rheumatoid arthritis. This review also provides the perspective that miRNA studies will identify not only new mechanisms in basic bone research, but also potential novel diagnostic biomarkers and drug targets for bone diseases.

Colorectal tumor molecular phenotype and miRNA: expression profiles and prognosis.

PubMed

Slattery, Martha L; Herrick, Jennifer S; Mullany, Lila E; Wolff, Erica; Hoffman, Michael D; Pellatt, Daniel F; Stevens, John R; Wolff, Roger K

2016-08-01

MiRNAs regulate gene expression by post-transcriptionally suppressing mRNA translation or by causing mRNA degradation. It has been proposed that unique miRNAs influence specific tumor molecular phenotype. In this paper, we test the hypotheses that miRNA expression differs by tumor molecular phenotype and that those differences may influence prognosis. Data come from population-based studies of colorectal cancer conducted in Utah and the Northern California Kaiser Permanente Medical Care Program. A total of 1893 carcinoma samples were run on the Agilent Human miRNA Microarray V19.0 containing 2006 miRNAs. We assessed differences in miRNA expression between TP53-mutated and non-mutated, KRAS-mutated and non-mutated, BRAF-mutated and non-mutated, CpG island methylator phenotype (CIMP) high and CIMP low, and microsatellite instability (MSI) and microsatellite stable (MSS) colon and rectal tumors. Using a Cox proportional hazard model we evaluated if those miRNAs differentially expressed by tumor phenotype influenced survival after adjusting for age, sex, and AJCC stage. There were 22 differentially expressed miRNAs for TP53-mutated colon tumors and 5 for TP53-mutated rectal tumors with a fold change of >1.49 (or <0.67). Additionally, 13 miRNAS were differentially expressed for KRAS-mutated rectal tumors, 8 differentially expressed miRNAs for colon CIMP high tumors, and 2 differentially expressed miRNAs for BRAF-mutated colon tumors. The majority of differentially expressed miRNAS were observed between MSI and MSS tumors (94 differentially expressed miRNAs for colon; 41 differentially expressed miRNAs for rectal tumors). Of these miRNAs differentially expressed between MSI and MSS tumors, the majority were downregulated. Ten of the differentially expressed miRNAs were associated with survival; after adjustment for MSI status, five miRNAS, miR-196b-5p, miR-31-5p, miR-99b-5p, miR-636, and miR-192-3p, were significantly associated with survival. In summary, it appears that the majority of miRNAs that are differentially expressed by tumor molecular phenotype are MSI tumors. However, these miRNAs appear to have minimal effect on prognosis.

Combination of miR-125b and miR-27a enhances sensitivity and specificity of AFP-based diagnosis of hepatocellular carcinoma.

PubMed

Zuo, Duo; Chen, Liwei; Liu, Xiaoqian; Wang, Xia; Xi, Qing; Luo, Yi; Zhang, Ning; Guo, Hua

2016-05-01

Non-invasive biomarkers of early-stage hepatocellular carcinoma (HCC) could offer immense benefits. Currently available tumor markers for HCC are of not much clinical relevance. In this study, we investigated the potential for using a panel of serum microRNAs (miRNAs) as novel tumor markers in conjunction with serum alpha-fetoprotein (AFP) for diagnosis of HCC. Serum expression of four miRNAs was assessed in 150 subjects (90 cases of HCC and 60 cases without cancer) by quantitative real-time polymerase chain reaction (qRT-PCR). Logistic regression analysis was performed to assess the potential use of miRNAs for detection of HCC. Receiver operating characteristic curves were used to evaluate diagnostic accuracy. A panel of serum miRNAs (miR-125b, miR-223, miR-27a, and miR-26a) used in conjunction with AFP helped differentiate HCC patients from those in the non-cancer group after adjusting for age and gender, with the area under the curve of 0.870. In addition, the use of miR-125b/miR-27a panel differentiated HBV-related early-stage HCC with a high sensitivity (80.0 %) and specificity (87.2 %) in AFP-negative (-) subjects. A combination of serum miR-125b, miR-223, miR-27a, and miR-26a as a second-line tests could help detect HCC in AFP (-) subjects. The panel of miR-125b/miR-27a/AFP had a higher sensitivity and specificity for diagnosis of early-stage HCC as compared to that of a single marker.

Conservation and divergence of microRNAs in Populus

PubMed Central

Barakat, Abdelali; Wall, Phillip K; DiLoreto, Scott; dePamphilis, Claude W; Carlson, John E

2007-01-01

Background MicroRNAs (miRNAs) are small RNAs (sRNA) ~21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in Arabidopsis and rice and partially investigated in other non-model plant species. To date, 109 and 62 miRNA families have been identified in Arabidopsis and rice respectively. However, only 33 miRNAs have been identified from the genome of the model tree species (Populus trichocarpa), of which 11 are Populus specific. The low number of miRNA families previously identified in Populus, compared with the number of families identified in Arabidopsis and rice, suggests that many miRNAs still remain to be discovered in Populus. In this study, we analyzed expressed small RNAs from leaves and vegetative buds of Populus using high throughput pyrosequencing. Results Analysis of almost eighty thousand small RNA reads allowed us to identify 123 new sequences belonging to previously identified miRNA families as well as 48 new miRNA families that could be Populus-specific. Comparison of the organization of miRNA families in Populus, Arabidopsis and rice showed that miRNA family sizes were generally expanded in Populus. The putative targets of non-conserved miRNA include both previously identified targets as well as several new putative target genes involved in development, resistance to stress, and other cellular processes. Moreover, almost half of the genes predicted to be targeted by non-conserved miRNAs appear to be Populus-specific. Comparative analyses showed that genes targeted by conserved and non-conserved miRNAs are biased mainly towards development, electron transport and signal transduction processes. Similar results were found for non-conserved miRNAs from Arabidopsis. Conclusion Our results suggest that while there is a conserved set of miRNAs among plant species, a large fraction of miRNAs vary among species. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to the taxa that produce them, as appears likely to be the case for those miRNAs that have only been observed in Populus. Non-conserved and conserved miRNAs seem to target genes with similar biological functions indicating that similar selection pressures are acting on both types of miRNAs. The expansion in the number of most conserved miRNAs in Populus relative to Arabidopsis, may be linked to the recent genome duplication in Populus, the slow evolution of the Populus genome, or to differences in the selection pressure on duplicated miRNAs in these species. PMID:18166134

LncRNAs expression in adjuvant-induced arthritis rats reveals the potential role of LncRNAs contributing to rheumatoid arthritis pathogenesis.

PubMed

Jiang, Hui; Qin, Xiu-Juan; Li, Wei-Ping; Ma, Rong; Wang, Ting; Li, Zhu-Qing

2016-11-15

Long non-coding RNAs (LncRNAs) are an important class of widespread molecules involved in diverse biological functions, which are exceptionally expressed in numerous types of diseases. Currently, limited study on LncRNA in rheumatoid arthritis (RA) is available. In this study, we aimed to identify the specifically expressed LncRNA that are relevant to adjuvant-induced arthritis (AA) in rats, and to explore the possible molecular mechanisms of RA pathogenesis. To identify LncRNAs specifically expressed in rheumatoid arthritis, the expression of LncRNAs in synoviums of rats from the model group (n=3) was compared with that in the control group (n=3) using Arraystar Rat LncRNA/mRNA microarray and real-time polymerase chain reaction (RT-PCR). Up to 260 LncRNAs were found to be differentially expressed (≥1.5-fold-change) in the synoviums between AA model and the normal rats (170 up-regulated and 90 down-regulated LncRNAs in AA rats compared with normal rats). Coding-non-coding gene co-expression networks (CNC network) were drawn based on the correlation analysis between the differentially expressed LncRNAs and mRNAs. Six LncRNAs, XR_008357, U75927, MRAK046251, XR_006457, DQ266363 and MRAK003448, were selected to analyze the relationship between LncRNAs and RA via the CNC network and GO analysis. Real-time PCR result confirmed that the six LncRNAs were specifically expressed in the AA rats. These results revealed that clusters of LncRNAs were uniquely expressed in AA rats compared with controls, which manifests that these differentially expressed LncRNAs in AA rats might play a vital role in RA development. Up-regulation or down-regulation of the six LncRNAs might contribute to the molecular mechanism underlying RA. To sum up, our study provides potential targets for treatment of RA and novel profound understanding of the pathogenesis of RA. Copyright © 2016. Published by Elsevier B.V.

Integrated Strategy Improves the Prediction Accuracy of miRNA in Large Dataset

PubMed Central

Lipps, David; Devineni, Sree

2016-01-01

MiRNAs are short non-coding RNAs of about 22 nucleotides, which play critical roles in gene expression regulation. The biogenesis of miRNAs is largely determined by the sequence and structural features of their parental RNA molecules. Based on these features, multiple computational tools have been developed to predict if RNA transcripts contain miRNAs or not. Although being very successful, these predictors started to face multiple challenges in recent years. Many predictors were optimized using datasets of hundreds of miRNA samples. The sizes of these datasets are much smaller than the number of known miRNAs. Consequently, the prediction accuracy of these predictors in large dataset becomes unknown and needs to be re-tested. In addition, many predictors were optimized for either high sensitivity or high specificity. These optimization strategies may bring in serious limitations in applications. Moreover, to meet continuously raised expectations on these computational tools, improving the prediction accuracy becomes extremely important. In this study, a meta-predictor mirMeta was developed by integrating a set of non-linear transformations with meta-strategy. More specifically, the outputs of five individual predictors were first preprocessed using non-linear transformations, and then fed into an artificial neural network to make the meta-prediction. The prediction accuracy of meta-predictor was validated using both multi-fold cross-validation and independent dataset. The final accuracy of meta-predictor in newly-designed large dataset is improved by 7% to 93%. The meta-predictor is also proved to be less dependent on datasets, as well as has refined balance between sensitivity and specificity. This study has two folds of importance: First, it shows that the combination of non-linear transformations and artificial neural networks improves the prediction accuracy of individual predictors. Second, a new miRNA predictor with significantly improved prediction accuracy is developed for the community for identifying novel miRNAs and the complete set of miRNAs. Source code is available at: https://github.com/xueLab/mirMeta PMID:28002428

Evidence for plant-derived xenomiRs based on a large-scale analysis of public small RNA sequencing data from human samples.

PubMed

Zhao, Qi; Liu, Yuanning; Zhang, Ning; Hu, Menghan; Zhang, Hao; Joshi, Trupti; Xu, Dong

2018-01-01

In recent years, an increasing number of studies have reported the presence of plant miRNAs in human samples, which resulted in a hypothesis asserting the existence of plant-derived exogenous microRNA (xenomiR). However, this hypothesis is not widely accepted in the scientific community due to possible sample contamination and the small sample size with lack of rigorous statistical analysis. This study provides a systematic statistical test that can validate (or invalidate) the plant-derived xenomiR hypothesis by analyzing 388 small RNA sequencing data from human samples in 11 types of body fluids/tissues. A total of 166 types of plant miRNAs were found in at least one human sample, of which 14 plant miRNAs represented more than 80% of the total plant miRNAs abundance in human samples. Plant miRNA profiles were characterized to be tissue-specific in different human samples. Meanwhile, the plant miRNAs identified from microbiome have an insignificant abundance compared to those from humans, while plant miRNA profiles in human samples were significantly different from those in plants, suggesting that sample contamination is an unlikely reason for all the plant miRNAs detected in human samples. This study also provides a set of testable synthetic miRNAs with isotopes that can be detected in situ after being fed to animals.

A pilot study of urinary microRNA as a biomarker for urothelial cancer

PubMed Central

Snowdon, Jaime; Boag, Sandy; Feilotter, Harriet; Izard, Jason; Siemens, D. Robert

2013-01-01

Objective: MicroRNAs (miRNAs) are part of a class of small ribonucleic acid (RNAs). They are important regulatory molecules, involved in several cell processes, such as developmental timing, stem cell division and apoptosis. Dysregulated miRNAs have been identified in several human malignancies, including bladder cancer tissue samples, and may confer a “tumour signature” that can be exploited for diagnostic purposes. We report on a prospective pilot study investigating the diagnostic capability of miRNAs in the urine of patients with urothelial cancer. Methods: Voided urine samples were collected from patients with urothelial carcinoma just prior to bladder tumour resection, as well as age-matched healthy control patients. Pathology demonstrated both low- and high-grade cancer. Total RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction was performed on the RNA extracts using primers for 4 miRNAs shown previously to be dysregulated in solid urothelial carcinomas with RNU6B as the endogenous control. Standard urine cytology was performed on all samples in a blinded fashion. Results: Two miRNAs of interest were dysregulated in the urine from cancer patients with miR-125b showing an average 10.42-fold decrease (p < 0.01) and miR-126 showing an average 2.70-fold increase (p = 0.30) in the cancer samples compared to the normal controls. The sensitivity and specificity of the cytology on the same urine samples were 50% and 80%, respectively. Using these 2 miRNAs only, a decision-tree prediction model was generated for a validation cohort of patients yielding a specificity of 100% and a sensitivity of 80%. Discussion: This preliminary study of candidate urinary miRNA in patients with low- and high-grade urothelial cancer demonstrated a significantly improved diagnostic accuracy over cytology. These results provide rationale for further studies on discovery and validation of candidate miRNAs in voided urine and may potentially lead to the development of a non-invasive and sensitive test for bladder cancer diagnosis and prognosis. PMID:22630336

Long noncoding RNAs and their proposed functions in fibre development of cotton (Gossypium spp.).

PubMed

Wang, Maojun; Yuan, Daojun; Tu, Lili; Gao, Wenhui; He, Yonghui; Hu, Haiyan; Wang, Pengcheng; Liu, Nian; Lindsey, Keith; Zhang, Xianlong

2015-09-01

Long noncoding RNAs (lncRNAs) are transcripts of at least 200 bp in length, possess no apparent coding capacity and are involved in various biological regulatory processes. Until now, no systematic identification of lncRNAs has been reported in cotton (Gossypium spp.). Here, we describe the identification of 30 550 long intergenic noncoding RNA (lincRNA) loci (50 566 transcripts) and 4718 long noncoding natural antisense transcript (lncNAT) loci (5826 transcripts). LncRNAs are rich in repetitive sequences and preferentially expressed in a tissue-specific manner. The detection of abundant genome-specific and/or lineage-specific lncRNAs indicated their weak evolutionary conservation. Approximately 76% of homoeologous lncRNAs exhibit biased expression patterns towards the At or Dt subgenomes. Compared with protein-coding genes, lncRNAs showed overall higher methylation levels and their expression was less affected by gene body methylation. Expression validation in different cotton accessions and coexpression network construction helped to identify several functional lncRNA candidates involved in cotton fibre initiation and elongation. Analysis of integrated expression from the subgenomes of lncRNAs generating miR397 and its targets as a result of genome polyploidization indicated their pivotal functions in regulating lignin metabolism in domesticated tetraploid cotton fibres. This study provides the first comprehensive identification of lncRNAs in Gossypium. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection.

PubMed

Kwenda, Stanford; Birch, Paul R J; Moleleki, Lucy N

2016-08-11

Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens.

Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs.

PubMed

Vyas, Meenal; Raza, Amir; Ali, Muhammad Yousaf; Ashraf, Muhammad Aleem; Mansoor, Shahid; Shahid, Ahmad Ali; Brown, Judith K

2017-01-01

Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly 'gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.

Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

PubMed

Morales-Prieto, D M; Ospina-Prieto, S; Schmidt, A; Chaiwangyen, W; Markert, U R

2014-02-01

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans. Copyright © 2013. Published by Elsevier Ltd.

First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection

PubMed Central

Fowler, Lauren; Conceicao, Viviane; Perera, Suneth S.; Gupta, Priyanka; Chew, Choo Beng; Dyer, Wayne B.; Saksena, Nitin K.

2016-01-01

The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART), therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV–) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV− samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV– controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs. PMID:29083374

Dual silencing of EGFR and HER2 enhances the sensitivity of gastric cancer cells to gefitinib.

PubMed

Wang, Liying; Zhang, Hongfeng; Zheng, Jiaxin; Wei, Xiaoli; Du, Jingwen; Lu, Haibo; Sun, Qiuying; Zhou, Weiyu; Zhang, Rui; Han, Yu

2018-04-10

Gefitinib exhibits very limited efficacy in gastric cancer (GC). Indeed, the limited clinical results obtained with gefitinib alone justify investigation of additional therapeutic strategies. Here, we demonstrate the importance of EGFR and HER2 in GC malignancy using RNA interference (RNAi). Additionally, we explored the ability of RNAi targeting EGFR and HER2 to enhance the sensitivity of GC cells to gefitinib. Specific small interfering RNAs (siRNAs) significantly inhibited mRNA and protein expression of target genes. EGFR-specific siRNA, EGFR/HER2 siRNAs, and gefitinib inhibited growth and induced apoptosis in GC cell lines in a dose-dependent manner. In contrast, resistance to HER2-siRNA-induced growth inhibition and apoptosis was linked to compensatory activation of EGFR. Moreover, gefitinib dramatically reduced p-EGFR and p-HER2 levels in the cell lines tested, and sensitivity to gefitinib was enhanced through dual silencing of EGFR and HER2 via suppression of AKT and ERK activation. These findings are in agreement with the profound inhibitory effect of gefitinib on activation of both EGFR and HER2. Overall, EGFR/HER2 knockdown by siRNAs further decreased the growth of GC cells treated with gefitinib alone, confirming that single-agent drug targeting does not achieve a maximal biological effect. The combination of gefitinib with EGFR/HER2 siRNAs should be further investigated as a new strategy for the treatment of GC and other EGFR/HER2-dependent cancers. © 2018 Wiley Periodicals, Inc.

Genome-wide maps of m6A circRNAs identify widespread and cell-type-specific methylation patterns that are distinct from mRNAs

PubMed Central

Zhou, Chan; Molinie, Benoit; Daneshvar, Kaveh; Pondick, Joshua V.; Wang, Jinkai; Van Wittenberghe, Nicholas O.; Xing, Yi; Giallourakis, Cosmas C.; Mullen, Alan C.

2017-01-01

Summary N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that together with depletion of ribosomal RNA and m6A immunoprecipitation defined thousands of m6A-circRNAs, with cell-type-specific expression. The presence of m6A-circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons that are not methylated in mRNAs, while mRNAs that are methylated on the same exons that compose m6A-circRNAs exhibit less stability, in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification. PMID:28854373

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

PubMed

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Bruhn, Laurakay; Dellinger, Douglas J

2018-01-25

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

PubMed Central

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Dellinger, Douglas J

2018-01-01

Abstract CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. PMID:29216382

Genome-Wide Analysis of miRNA targets in Brachypodium and Biomass Energy Crops

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, Pamela J.

2015-08-11

MicroRNAs (miRNAs) contribute to the control of numerous biological processes through the regulation of specific target mRNAs. Although the identities of these targets are essential to elucidate miRNA function, the targets are much more difficult to identify than the small RNAs themselves. Before this work, we pioneered the genome-wide identification of the targets of Arabidopsis miRNAs using an approach called PARE (German et al., Nature Biotech. 2008; Nature Protocols, 2009). Under this project, we applied PARE to Brachypodium distachyon (Brachypodium), a model plant in the Poaceae family, which includes the major food grain and bioenergy crops. Through in-depth global analysismore » and examination of specific examples, this research greatly expanded our knowledge of miRNAs and target RNAs of Brachypodium. New regulation in response to environmental stress or tissue type was found, and many new miRNAs were discovered. More than 260 targets of new and known miRNAs with PARE sequences at the precise sites of miRNA-guided cleavage were identified and characterized. Combining PARE data with the small RNA data also identified the miRNAs responsible for initiating approximately 500 phased loci, including one of the novel miRNAs. PARE analysis also revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. The project included generation of small RNA and PARE resources for bioenergy crops, to facilitate ongoing discovery of conserved miRNA-target RNA regulation. By associating specific miRNA-target RNA pairs with known physiological functions, the research provides insights about gene regulation in different tissues and in response to environmental stress. This, and release of new PARE and small RNA data sets should contribute basic knowledge to enhance breeding and may suggest new strategies for improvement of biomass energy crops.« less

The Big Entity of New RNA World: Long Non-Coding RNAs in Microvascular Complications of Diabetes.

PubMed

Raut, Satish K; Khullar, Madhu

2018-01-01

A major part of the genome is known to be transcribed into non-protein coding RNAs (ncRNAs), such as microRNA and long non-coding RNA (lncRNA). The importance of ncRNAs is being increasingly recognized in physiological and pathological processes. lncRNAs are a novel class of ncRNAs that do not code for proteins and are important regulators of gene expression. In the past, these molecules were thought to be transcriptional "noise" with low levels of evolutionary conservation. However, recent studies provide strong evidence indicating that lncRNAs are (i) regulated during various cellular processes, (ii) exhibit cell type-specific expression, (iii) localize to specific organelles, and (iv) associated with human diseases. Emerging evidence indicates an aberrant expression of lncRNAs in diabetes and diabetes-related microvascular complications. In the present review, we discuss the current state of knowledge of lncRNAs, their genesis from genome, and the mechanism of action of individual lncRNAs in the pathogenesis of microvascular complications of diabetes and therapeutic approaches.

Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation

PubMed Central

Warth, Sebastian C; Hoefig, Kai P; Hiekel, Anian; Schallenberg, Sonja; Jovanovic, Ksenija; Klein, Ludger; Kretschmer, Karsten; Ansel, K Mark; Heissmeyer, Vigo

2015-01-01

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4+ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4+ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs. PMID:25712478

Specificity Protein (Sp) Transcription Factors and Metformin Regulate Expression of the Long Non-coding RNA HULC

EPA Science Inventory

There is evidence that specificity protein 1 (Sp1) transcription factor (TF) regulates expression of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) cells. RNA interference (RNAi) studies showed that among several lncRNAs expressed in HepG2, SNU-449 and SK-Hep-1...

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells

PubMed Central

Hyatt, Sam; Cheung, Kat; Skelton, Andrew J.; Xu, Yaobo; Clark, Ian M.

2017-01-01

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA. PMID:29084806

Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis.

PubMed

Pan, Ting; Sun, Xiuqiang; Liu, Yangxuan; Li, Hui; Deng, Guangbin; Lin, Honghui; Wang, Songhu

2018-02-01

1599 novel circRNAs and 1583 heat stress-specific circRNAs were identified in Arabidopsis. Heat stress enhanced accumulation of circRNAs remarkably. Heat stress altered the sizes of circRNAs, numbers of circularized exons and alterative circularization events. A putative circRNA-mediated ceRNA networks under heat stress was established. Heat stress retards plant growth and destabilizes crop yield. The noncoding RNAs were demonstrated to be involved in plant response to heat stress. As a newly-characterized class of noncoding RNAs, circular RNAs (circRNAs) play important roles in transcriptional and post-transcriptional regulation. A few recent investigations indicated that plant circRNAs were differentially expressed under abiotic stress. However, little is known about how heat stress mediates biogenesis of circRNAs in plants. Here, we uncovered 1599 previously-unknown circRNAs and 1583 heat-specific circRNAs, by RNA-sequencing and bioinformatic analysis. Our results indicated that much more circRNAs were expressed under heat stress than in control condition. Besides, heat stress also increased the length of circRNAs, the quantity of circularized exons, and alternative circularization events. Moreover, we observed a positive correlation between expression patterns of some circRNAs and their parental genes. The prediction of ceRNA (competing endogenous RNA) networks indicated that differentially-expressed circRNAs could influence expression of many important genes, that participate in response to heat stress, hydrogen peroxide, and phytohormone signaling pathways, by interacting with the corresponding microRNAs. Together, our observations indicated that heat stress had great impacts on the biogenesis of circRNAs. Heat-induced circRNAs might participate in plant response to heat stress through the circRNA-mediated ceRNA networks.

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling

PubMed Central

Lin, Wan-Hsin; Lu, Ruifeng; Feathers, Ryan W.; Asmann, Yan W.; Thompson, E. Aubrey

2017-01-01

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis. PMID:28877994

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling.

PubMed

Kourtidis, Antonis; Necela, Brian; Lin, Wan-Hsin; Lu, Ruifeng; Feathers, Ryan W; Asmann, Yan W; Thompson, E Aubrey; Anastasiadis, Panos Z

2017-10-02

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis. © 2017 Kourtidis et al.

The expanding roles of microRNAs in kidney pathophysiology.

PubMed

Metzinger-Le Meuth, Valérie; Fourdinier, Ophélie; Charnaux, Nathalie; Massy, Ziad A; Metzinger, Laurent

2018-05-25

MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3'-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system.

The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

PubMed

Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

2016-01-01

MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

Identification and Characterization of MicroRNAs in Ovary and Testis of Nile Tilapia (Oreochromis niloticus) by Using Solexa Sequencing Technology

PubMed Central

Zhou, Yi; Yu, Fan; Gao, Yun; Luo, Yongju; Tang, Zhanyang; Guo, Zhongbao; Guo, Enyan; Gan, Xi; Zhang, Ming; Zhang, Yaping

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs which play important roles in the regulation of gene expression by cleaving or inhibiting the translation of target gene transcripts. Thereinto, some specific miRNAs show regulatory activities in gonad development via translational control. In order to further understand the role of miRNA-mediated posttranscriptional regulation in Nile tilapia (Oreochromis niloticus) ovary and testis, two small RNA libraries of Nile tilapia were sequenced by Solexa small RNA deep sequencing methods. A total of 9,731,431 and 8,880,497 raw reads, representing 5,407,800 and 4,396,281 unique sequences were obtained from the sexually mature ovaries and testes, respectively. After comparing the small RNA sequences with the Rfam database, 1,432,210 reads in ovaries and 984,146 reads in testes were matched to the genome sequence of Nile tilapia. Bioinformatic analysis identified 764 mature miRNA, 209 miRNA-5p and 202 miRNA-3p were found in the two libraries, of which 525 known miRNAs are both expressed in the ovary and testis of Nile tilapia. Comparison of expression profiles of the testis, miR-727, miR-129 and miR-29 families were highly expressed in tilapia ovary. Additionally, miR-132, miR-212, miR-33a and miR-135b families, showed significant higher expression in testis compared with that in ovary. Furthermore, the expression patterns of the miRNAs were analyzed in different developmental stages of gonad. The result showed different expression patterns were observed during development of testis and ovary. In addition, the identification and characterization of differentially expressed miRNAs in the ovaries and testis of Nile tilapia provides important information on the role of miRNA in the regulation of the ovarian and testicular development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during teleosts reproduction. PMID:24466258

Sexual Dimorphism Floral MicroRNA Profiling and Target Gene Expression in Andromonoecious Poplar (Populus tomentosa)

PubMed Central

Song, Yuepeng; Ma, Kaifeng; Ci, Dong; Zhang, Zhiyi; Zhang, Deqiang

2013-01-01

Although the molecular basis of poplar sex-specific flower development remains largely unknown, increasing evidence indicates an essential role for microRNAs (miRNAs). The specific miRNA types and precise miRNA expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. This system, combined with high-throughput sequencing and computational analysis, allowed us to characterize sex-specific miRNAomes from female and male flowers. Comparative miRNAome analysis combined with quantitative real-time PCR revealed the expression patterns of 27 miRNAs in poplar flower and showed that the targets of these miRNAs are involved in flower organogenesis, Ca2+ transport, phytohormone synthesis and metabolism, and DNA methylation. This paper describes a complex regulatory network consisting of these miRNAs expressed in sex-specific flower development in a dioecious plant. The conserved and novel miRNA locations were annotated in the Populus trichocarpa genome. Among these, miRNA Pto-F70 and 4 targets are located in the sex-determination regions of chromosome XIX. Furthermore, two novel miRNAs, Pto-F47 and Pto-F68, were shown for the first time to be regulatory factors in phytohormone interactions. To our knowledge, this report is the first systematic investigation of sex-specific flower-related miRNAs and their targets in poplar, and it deepens our understanding of the important regulatory functions of miRNAs in female and male flower development in this dioecious plant. PMID:23667507

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

PubMed

Pu, Junhua; Li, Rui; Zhang, Chenglong; Chen, Dan; Liao, Xiangxiang; Zhu, Yihui; Geng, Xiaohan; Ji, Dejun; Mao, Yongjiang; Gong, Yunchen; Yang, Zhangping

2017-08-01

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

PubMed

Lueong, Smiths; Leong, Smiths; Simo, Gustave; Camara, Mamadou; Jamonneau, Vincent; Kabore, Jacques; Ilboudo, Hamidou; Bucheton, Bruno; Hoheisel, Jörg D; Clayton, Christine

2013-01-01

Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II) and without (stage I) brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II), 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested) showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

Phytoalexins, miRNAs and breast cancer: a review of phytochemical mediated miRNA regulation in breast cancer

USDA-ARS?s Scientific Manuscript database

A specific class of endogenous, non-coding RNAs, classified as microRNAs (miRNAs), has been identified. It has been found that miRNAs are associated with many biological processes and disease states, including all stages of cancer from initiation to tumor promotion and progression. These studies d...

Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and miRNAs directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolate...

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

miRNAs in brain development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petri, Rebecca; Malmevik, Josephine; Fasching, Liana

2014-02-01

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs havemore » been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function.« less

mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

NASA Astrophysics Data System (ADS)

Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

2016-11-01

Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum.

PubMed

Yu, Huilin; Cong, Ling; Zhu, Zhenxing; Wang, Chunyu; Zou, Jianqiu; Tao, Chengguang; Shi, Zhensheng; Lu, Xiaochun

2015-10-25

MicroRNAs (miRNAs) have been shown to play important roles in plant development, growth and stress response. Sweet sorghum [Sorghum bicolor (L.) Moench] is an important source of bioenergy due to the high sugar content in its stems. However, it is not clear how the miRNA is involved in sugar accumulation in sorghum stems. In order to identify the miRNAs in the stems and the leaves of sweet sorghum, we extracted RNAs of the stems and leaves of sweet sorghum (Rio) and grain sorghum (BTx623) at the heading and dough stages for high-throughput sequencing. A total of 179279048 reads were obtained from Illumina-based sequencing. Further analysis identified nine known miRNAs and twelve novel miRNAs that showed significantly and specifically differentially expressed in the stems of sweet sorghum. The target genes of the differentially expressed novel miRNAs include the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporters etc. GO enrichment analysis showed that the predicted targets of these differentially expressed miRNAs participated in diverse physiological and metabolic processes. We performed RT-qRCR analysis on these miRNAs across eight different libraries to validate the miRNAs. Finally, we screened stem-specifically expressed novel miRNA and a leaf-specifically expressed novel miRNA in sweet sorghum comparing with grain sorghum. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

PubMed

Yerramilli, V Siddartha; Kim, Kyung Hyuk

2018-03-16

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

Detection and quantification of extracellular microRNAs in murine biofluids

PubMed Central

2014-01-01

Background MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. Results The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used. Conclusions Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity. PMID:24629058

Genome-wide identification of long non-coding RNA genes and their association with insecticide resistance and metamorphosis in diamondback moth, Plutella xylostella.

PubMed

Liu, Feiling; Guo, Dianhao; Yuan, Zhuting; Chen, Chen; Xiao, Huamei

2017-11-20

Long non-coding RNA (lncRNA) is a class of noncoding RNA >200 bp in length that has essential roles in regulating a variety of biological processes. Here, we constructed a computational pipeline to identify lncRNA genes in the diamondback moth (Plutella xylostella), a major insect pest of cruciferous vegetables. In total, 3,324 lncRNAs corresponding to 2,475 loci were identified from 13 RNA-Seq datasets, including samples from parasitized, insecticide-resistant strains and different developmental stages. The identified P. xylostella lncRNAs had shorter transcripts and fewer exons than protein-coding genes. Seven out of nine randomly selected lncRNAs were validated by strand-specific RT-PCR. In total, 54-172 lncRNAs were specifically expressed in the insecticide resistant strains, among which one lncRNA was located adjacent to the sodium channel gene. In addition, 63-135 lncRNAs were specifically expressed in different developmental stages, among which three lncRNAs overlapped or were located adjacent to the metamorphosis-associated genes. These lncRNAs were either strongly or weakly co-expressed with their overlapping or neighboring mRNA genes. In summary, we identified thousands of lncRNAs and presented evidence that lncRNAs might have key roles in conferring insecticide resistance and regulating the metamorphosis development in P. xylostella.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

PubMed Central

Kozhuharova, Ana; Sharma, Harshita; Ohyama, Takako; Fasolo, Francesca; Yamazaki, Toshio; Cotella, Diego; Santoro, Claudio; Zucchelli, Silvia; Gustincich, Stefano; Carninci, Piero

2018-01-01

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest. PMID:29414979

Identification of the miRNA targetome in hippocampal neurons using RIP-seq.

PubMed

Malmevik, Josephine; Petri, Rebecca; Klussendorf, Thies; Knauff, Pina; Åkerblom, Malin; Johansson, Jenny; Soneji, Shamit; Jakobsson, Johan

2015-07-28

MicroRNAs (miRNAs) are key players in the regulation of neuronal processes by targeting a large network of target messenger RNAs (mRNAs). However, the identity and function of mRNAs targeted by miRNAs in specific cells of the brain are largely unknown. Here, we established an adeno-associated viral vector (AAV)-based neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this approach, we identified more than two thousand miRNA targets in hippocampal neurons, regulating essential neuronal features such as cell signalling, transcription and axon guidance. Furthermore, we found that stable inhibition of the highly expressed miR-124 and miR-125 in hippocampal neurons led to significant but distinct changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. These findings greatly enhance our understanding of the miRNA targetome in hippocampal neurons.

A discovery of novel microRNAs in the silkworm (Bombyx mori) genome.

PubMed

Yu, Xiaomin; Zhou, Qing; Cai, Yimei; Luo, Qibin; Lin, Hongbin; Hu, Songnian; Yu, Jun

2009-12-01

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

Circular RNAs and hereditary bone diseases.

PubMed

Zhai, Naixiang; Lu, Yanqin; Wang, Yanzhou; Ren, Xiuzhi; Han, Jinxiang

2018-02-01

Circular RNA (circRNA) is a non-linear form of RNA derived from exonic, intronic, and exon-intron gene regions. circRNAs are characterized by covalent closed loops, highly stable nuclease resistance, and specific expression in species and developmental stages. CircRNA molecules have been identified as playing roles in the regulation of cell transcription, transcriptional expression after translation, interactions with microRNAs, and protein coding. A high stability and tissue- and disease-specific expression allow circRNAs to serve as potential biomarkers both for diseases and prognosis. CircRNAs function in bone remodeling by directly participating in bone-related signaling pathways and by forming the circRNA-miRNA-mRNA axis. Studies have seldom reported on the low incidence of circRNAs in genetic bone disorders. The current study reviews the characteristics of circRNAs and recent research on their role in rare hereditary bone diseases.

Non-coding RNAs: new biomarkers and therapeutic targets for esophageal cancer

PubMed Central

Ren, Zhipeng; Zhang, Guoliang

2017-01-01

Esophageal cancer is one of the most common gastrointestinal malignant diseases and there is still no effective treatment. The incidence of esophageal cancer in the world is relatively high and on the increase year by year. Thus, the elaboration on the carcinogenesis of esophageal cancer and the identification of new biomarkers and therapeutic targets is quite beneficial to optimizing the current therapeutic regimen for treating such deadly disease. More and more evidence has shown that non-coding RNAs play an important role in the development and progression of multiple human cancers, including esophageal cancer. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two functional kinds of non-coding RNAs that have been well investigated. They exert tumor suppressive or promoting effect by specifically regulating the expression of certain downstream target genes, which is tumor specific. It is also proved that miRNAs and lncRNAs level in tissue and plasma from esophageal cancer patients are closely correlated with the survival and disease progression, which could be used as a prognostic factor and therapeutic target for esophageal cancer. PMID:28388588

Non-coding RNAs: new biomarkers and therapeutic targets for esophageal cancer.

PubMed

Hou, Xiaobin; Wen, Jiaxin; Ren, Zhipeng; Zhang, Guoliang

2017-06-27

Esophageal cancer is one of the most common gastrointestinal malignant diseases and there is still no effective treatment. The incidence of esophageal cancer in the world is relatively high and on the increase year by year. Thus, the elaboration on the carcinogenesis of esophageal cancer and the identification of new biomarkers and therapeutic targets is quite beneficial to optimizing the current therapeutic regimen for treating such deadly disease. More and more evidence has shown that non-coding RNAs play an important role in the development and progression of multiple human cancers, including esophageal cancer. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two functional kinds of non-coding RNAs that have been well investigated. They exert tumor suppressive or promoting effect by specifically regulating the expression of certain downstream target genes, which is tumor specific. It is also proved that miRNAs and lncRNAs level in tissue and plasma from esophageal cancer patients are closely correlated with the survival and disease progression, which could be used as a prognostic factor and therapeutic target for esophageal cancer.

Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity.

PubMed

Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

2014-01-01

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes

PubMed Central

Watanabe, Toshiaki; Takeda, Atsushi; Tsukiyama, Tomoyuki; Mise, Kazuyuki; Okuno, Tetsuro; Sasaki, Hiroyuki; Minami, Naojiro; Imai, Hiroshi

2006-01-01

Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts, plants, and animals. Small RNAs are involved in the regulation of gene expression through translational repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in the mouse germline. One class consists of ∼20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes, which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of retrotransposon-derived sequences to the 3′ untranslated region (UTR) of a reporter mRNA destabilizes the mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These features are reminiscent of Tetrahymena ∼23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster small RNAs. A conserved novel small RNA pathway may be present in diverse animals. PMID:16766679

miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis.

PubMed

Creasey, Kate M; Zhai, Jixian; Borges, Filipe; Van Ex, Frederic; Regulski, Michael; Meyers, Blake C; Martienssen, Robert A

2014-04-17

In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence.

Inhibition of atherosclerosis-promoting microRNAs via targeted polyelectrolyte complex micelles

PubMed Central

Kuo, Cheng-Hsiang; Leon, Lorraine; Chung, Eun Ji; Huang, Ru-Ting; Sontag, Timothy J.; Reardon, Catherine A.; Getz, Godfrey S.; Tirrell, Matthew; Fang, Yun

2015-01-01

Polyelectrolyte complex micelles have great potential as gene delivery vehicles because of their ability to encapsulate charged nucleic acids forming a core by neutralizing their charge, while simultaneously protecting the nucleic acids from non-specific interactions and enzymatic degradation. Furthermore, to enhance specificity and transfection efficiency, polyelectrolyte complex micelles can be modified to include targeting capabilities. Here, we describe the design of targeted polyelectrolyte complex micelles containing inhibitors against dys-regulated microRNAs (miRNAs) that promote atherosclerosis, a leading cause of human mortality and morbidity. Inhibition of dys-regulated miRNAs in diseased cells associated with atherosclerosis has resulted in therapeutic efficacy in animal models and has been proposed to treat human diseases. However, the non-specific targeting of microRNA inhibitors via systemic delivery has remained an issue that may cause unwanted side effects. For this reason, we incorporated two different peptide sequences to our miRNA inhibitor containing polyelectrolyte complex micelles. One of the peptides (Arginine-Glutamic Acid-Lysine-Alanine or REKA) was used in another micellar system that demonstrated lesion-specific targeting in a mouse model of atherosclerosis. The other peptide (Valine-Histidine-Proline-Lysine-Glutamine-Histidine-Arginine or VHPKQHR) was identified via phage display and targets vascular endothelial cells through the vascular cell adhesion molecule-1 (VCAM-1). In this study we have tested the in vitro efficacy and efficiency of lesion- and cell-specific delivery of microRNA inhibitors to the cells associated with atherosclerotic lesions via peptide-targeted polyelectrolyte complex micelles. Our results show that REKA-containing micelles (fibrin-targeting) and VHPKQHR-containing micelles (VCAM-1 targeting) can be used to carry and deliver microRNA inhibitors into macrophages and human endothelial cells, respectively. Additionally, the functionality of miRNA inhibitors in cells was demonstrated by analyzing miRNA expression as well as the expression or the biological function of its downstream target protein. Our study provides the first demonstration of targeting dys-regulated miRNAs in atherosclerosis using targeted polyelectrolyte complex micelles and holds promising potential for translational applications. PMID:25685357

Improved Discovery of Molecular Interactions in Genome-Scale Data with Adaptive Model-Based Normalization

PubMed Central

Brown, Patrick O.

2013-01-01

Background High throughput molecular-interaction studies using immunoprecipitations (IP) or affinity purifications are powerful and widely used in biology research. One of many important applications of this method is to identify the set of RNAs that interact with a particular RNA-binding protein (RBP). Here, the unique statistical challenge presented is to delineate a specific set of RNAs that are enriched in one sample relative to another, typically a specific IP compared to a non-specific control to model background. The choice of normalization procedure critically impacts the number of RNAs that will be identified as interacting with an RBP at a given significance threshold – yet existing normalization methods make assumptions that are often fundamentally inaccurate when applied to IP enrichment data. Methods In this paper, we present a new normalization methodology that is specifically designed for identifying enriched RNA or DNA sequences in an IP. The normalization (called adaptive or AD normalization) uses a basic model of the IP experiment and is not a variant of mean, quantile, or other methodology previously proposed. The approach is evaluated statistically and tested with simulated and empirical data. Results and Conclusions The adaptive (AD) normalization method results in a greatly increased range in the number of enriched RNAs identified, fewer false positives, and overall better concordance with independent biological evidence, for the RBPs we analyzed, compared to median normalization. The approach is also applicable to the study of pairwise RNA, DNA and protein interactions such as the analysis of transcription factors via chromatin immunoprecipitation (ChIP) or any other experiments where samples from two conditions, one of which contains an enriched subset of the other, are studied. PMID:23349766

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

PubMed

Kriegel, Alison J; Liu, Yong; Liu, Pengyuan; Baker, Maria Angeles; Hodges, Matthew R; Hua, Xing; Liang, Mingyu

2013-12-01

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera.

PubMed

Jayakodi, Murukarthick; Jung, Je Won; Park, Doori; Ahn, Young-Joon; Lee, Sang-Choon; Shin, Sang-Yoon; Shin, Chanseok; Yang, Tae-Jin; Kwon, Hyung Wook

2015-09-04

Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases. In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses. This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Surprisal analysis of Glioblastoma Multiform (GBM) microRNA dynamics unveils tumor specific phenotype.

PubMed

Zadran, Sohila; Remacle, Francoise; Levine, Raphael

2014-01-01

Gliomablastoma multiform (GBM) is the most fatal form of all brain cancers in humans. Currently there are limited diagnostic tools for GBM detection. Here, we applied surprisal analysis, a theory grounded in thermodynamics, to unveil how biomolecule energetics, specifically a redistribution of free energy amongst microRNAs (miRNAs), results in a system deviating from a non-cancer state to the GBM cancer -specific phenotypic state. Utilizing global miRNA microarray expression data of normal and GBM patients tumors, surprisal analysis characterizes a miRNA system response capable of distinguishing GBM samples from normal tissue biopsy samples. We indicate that the miRNAs contributing to this system behavior is a disease phenotypic state specific to GBM and is therefore a unique GBM-specific thermodynamic signature. MiRNAs implicated in the regulation of stochastic signaling processes crucial in the hallmarks of human cancer, dominate this GBM-cancer phenotypic state. With this theory, we were able to distinguish with high fidelity GBM patients solely by monitoring the dynamics of miRNAs present in patients' biopsy samples. We anticipate that the GBM-specific thermodynamic signature will provide a critical translational tool in better characterizing cancer types and in the development of future therapeutics for GBM.

An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1

PubMed Central

2010-01-01

Background HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases. Methods A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings. Results Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing. Conclusions HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted under specific antiretroviral selective pressure, but in both cases these should be tested exhaustively prior to clinical use. PMID:21172023

Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing.

PubMed

Yuan, Chao; Wang, Xiaolong; Geng, Rongqing; He, Xiaolin; Qu, Lei; Chen, Yulin

2013-07-28

MicroRNAs (miRNAs) are a large family of endogenous, non-coding RNAs, about 22 nucleotides long, which regulate gene expression through sequence-specific base pairing with target mRNAs. Extensive studies have shown that miRNA expression in the skin changes remarkably during distinct stages of the hair cycle in humans, mice, goats and sheep. In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fibre-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalisation analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. The expression level of five arbitrarily selected miRNAs was analyzed by quantitative PCR, and the results indicated that the expression patterns were consistent with the Solexa sequencing results. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounted for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling. During all hair cycle stages of cashmere goats, a large number of conserved and novel miRNAs were identified through a high-throughput sequencing approach. This study enriches the Capra hircus miRNA databases and provides a comprehensive miRNA transcriptome profile in the skin of goats during the hair follicle cycle.

Circulating Organ-Specific MicroRNAs Serve as Biomarkers in Organ-Specific Diseases: Implications for Organ Allo- and Xeno-Transplantation

PubMed Central

Zhou, Ming; Hara, Hidetaka; Dai, Yifan; Mou, Lisha; Cooper, David K. C.; Wu, Changyou; Cai, Zhiming

2016-01-01

Different cell types possess different miRNA expression profiles, and cell/tissue/organ-specific miRNAs (or profiles) indicate different diseases. Circulating miRNA is either actively secreted by living cells or passively released during cell death. Circulating cell/tissue/organ-specific miRNA may serve as a non-invasive biomarker for allo- or xeno-transplantation to monitor organ survival and immune rejection. In this review, we summarize the proof of concept that circulating organ-specific miRNAs serve as non-invasive biomarkers for a wide spectrum of clinical organ-specific manifestations such as liver-related disease, heart-related disease, kidney-related disease, and lung-related disease. Furthermore, we summarize how circulating organ-specific miRNAs may have advantages over conventional methods for monitoring immune rejection in organ transplantation. Finally, we discuss the implications and challenges of applying miRNA to monitor organ survival and immune rejection in allo- or xeno-transplantation. PMID:27490531

The developmental transcriptome of the mosquito Aedes aegypti, an invasive species and major arbovirus vector.

PubMed

Akbari, Omar S; Antoshechkin, Igor; Amrhein, Henry; Williams, Brian; Diloreto, Race; Sandler, Jeremy; Hay, Bruce A

2013-09-04

Mosquitoes are vectors of a number of important human and animal diseases. The development of novel vector control strategies requires a thorough understanding of mosquito biology. To facilitate this, we used RNA-seq to identify novel genes and provide the first high-resolution view of the transcriptome throughout development and in response to blood feeding in a mosquito vector of human disease, Aedes aegypti, the primary vector for Dengue and yellow fever. We characterized mRNA expression at 34 distinct time points throughout Aedes development, including adult somatic and germline tissues, by using polyA+ RNA-seq. We identify a total of 14,238 novel new transcribed regions corresponding to 12,597 new loci, as well as many novel transcript isoforms of previously annotated genes. Altogether these results increase the annotated fraction of the transcribed genome into long polyA+ RNAs by more than twofold. We also identified a number of patterns of shared gene expression, as well as genes and/or exons expressed sex-specifically or sex-differentially. Expression profiles of small RNAs in ovaries, early embryos, testes, and adult male and female somatic tissues also were determined, resulting in the identification of 38 new Aedes-specific miRNAs, and ~291,000 small RNA new transcribed regions, many of which are likely to be endogenous small-interfering RNAs and Piwi-interacting RNAs. Genes of potential interest for transgene-based vector control strategies also are highlighted. Our data have been incorporated into a user-friendly genome browser located at www.Aedes.caltech.edu, with relevant links to Vectorbase (www.vectorbase.org).

Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

PubMed

Calliari, Aldo; Farías, Joaquina; Puppo, Agostina; Canclini, Lucía; Mercer, John A; Munroe, David; Sotelo, José R; Sotelo-Silveira, José R

2014-03-01

Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems. Copyright © 2013 Wiley Periodicals, Inc.

Cellular and extracellular miRNAs are blood-compartment-specific diagnostic targets in sepsis.

PubMed

Reithmair, Marlene; Buschmann, Dominik; Märte, Melanie; Kirchner, Benedikt; Hagl, Daniel; Kaufmann, Ines; Pfob, Martina; Chouker, Alexander; Steinlein, Ortrud K; Pfaffl, Michael W; Schelling, Gustav

2017-10-01

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High-throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood-compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next-generation sequencing and RT-qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment-specific signalling functions of differentially regulated miRNAs in sepsis-relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down- and up-regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment-specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR-199b-5p was identified as a potential early indicator for sepsis and septic shock. miR-125b-5p and miR-26b-5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR-27b-3p) was present in all three compartments. The expression of sepsis-associated miRNAs is compartment-specific. Exosome-derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Genome-wide identification of Hami melon miRNAs with putative roles during fruit development

PubMed Central

Wang, Guangzhi; Ma, Xinli; Li, Meihua; Wu, Haibo; Fu, Qiushi; Zhang, Yi; Yi, Hongping

2017-01-01

MicroRNAs represent a family of small endogenous, non-coding RNAs that play critical regulatory roles in plant growth, development, and environmental stress responses. Hami melon is famous for its attractive flavor and excellent nutritional value, however, the mechanisms underlying the fruit development and ripening remains largely unknown. Here, we performed small RNA sequencing to investigate the roles of miRNAs during Hami melon fruit development. Two batches of flesh samples were collected at four fruit development stages. Small RNA sequencing yielded a total of 54,553,424 raw reads from eight libraries. 113 conserved miRNAs belonging to 30 miRNA families and nine novel miRNAs comprising nine miRNA families were identified. The expression of 42 conserved miRNAs and three Hami melon-specific miRNAs significantly changed during fruit development. Furthermore, 484 and 124 melon genes were predicted as putative targets of 29 conserved and nine Hami melon-specific miRNA families, respectively. GO enrichment analysis were performed on target genes, “transcription, DNA-dependent”, “rRNA processing”, “oxidation reduction”, “signal transduction”, “regulation of transcription, DNA-dependent”, and “metabolic process” were the over-represented biological process terms. Cleavage sites of six target genes were validated using 5’ RACE. Our results present a comprehensive set of identification and characterization of Hami melon fruit miRNAs and their potential targets, which provide valuable basis towards understanding the regulatory mechanisms in programmed process of normal Hami fruit development and ripening. Specific miRNAs could be selected for further research and applications in breeding practices. PMID:28742088

Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants.

PubMed

Palukaitis, P; García-Arenal, F; Sulzinski, M A; Zaitlin, M

1983-12-01

The single-stranded (ss) and double-stranded (ds) RNAs produced in tobacco tissue as a result of infection by tobacco mosaic virus (TMV) have been reinvestigated. 32P-labeled probes consisting of either cDNA or viral RNA, complementary to specific regions of either the viral RNA or its negative strand, respectively, were used in "Northern" hybridization experiments. Of the 10 ssRNA bands observed, all but four appeared to be artifacts of electrophoresis. These four RNAs were found on polyribosomes and are presumed to be true mRNAs; three were identified as the well-known genomic RNA, the I2-mRNA and the coat protein mRNA, or LMC. The fourth RNA species of MW approximately 1.2 x 10(6) had not previously been specifically identified as a subgenomic RNA of TMV. The viral RNA which gave rise to the six artifactual ssRNA bands was heterogeneous in size and was shown to be encapsidated in vivo. Upon electrophoresis, these heterogeneous RNA fragments comigrated approximately with plant rRNAs also present in the extracts, generating the observed artifactual bands. Four dsRNAs were also identified. From molecular weight and hybridization analyses, they appeared to be double-stranded forms of the above four polyribosome-associated ssRNAs. Attempts to translate proteins from the denatured dsRNAs in vitro were unsuccessful. A population of low-molecular-weight, TMV-specific ssRNAs, (+) and (-) in sequence, was generated during infection; however these RNAs were believed to be breakdown products.

Plant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees.

PubMed

Williams, Philip H; Eyles, Rod; Weiller, Georg

2012-01-01

MicroRNAs (miRNAs) are nonprotein coding RNAs between 20 and 22 nucleotides long that attenuate protein production. Different types of sequence data are being investigated for novel miRNAs, including genomic and transcriptomic sequences. A variety of machine learning methods have successfully predicted miRNA precursors, mature miRNAs, and other nonprotein coding sequences. MirTools, mirDeep2, and miRanalyzer require "read count" to be included with the input sequences, which restricts their use to deep-sequencing data. Our aim was to train a predictor using a cross-section of different species to accurately predict miRNAs outside the training set. We wanted a system that did not require read-count for prediction and could therefore be applied to short sequences extracted from genomic, EST, or RNA-seq sources. A miRNA-predictive decision-tree model has been developed by supervised machine learning. It only requires that the corresponding genome or transcriptome is available within a sequence window that includes the precursor candidate so that the required sequence features can be collected. Some of the most critical features for training the predictor are the miRNA:miRNA(∗) duplex energy and the number of mismatches in the duplex. We present a cross-species plant miRNA predictor with 84.08% sensitivity and 98.53% specificity based on rigorous testing by leave-one-out validation.

Differential blood-based diagnosis between benign prostatic hyperplasia and prostate cancer: miRNA as source for biomarkers independent of PSA level, Gleason score, or TNM status.

PubMed

Leidinger, Petra; Hart, Martin; Backes, Christina; Rheinheimer, Stefanie; Keck, Bastian; Wullich, Bernd; Keller, Andreas; Meese, Eckart

2016-08-01

Since the benefit of prostate-specific antigen (PSA) screening remains controversial, new non-invasive biomarkers for prostate carcinoma (PCa) are still required. There is evidence that microRNAs (miRNAs) in whole peripheral blood can separate patients with localized prostate cancer from healthy individuals. However, the potential of blood-based miRNAs for the differential diagnosis of PCa and benign prostatic hyperplasia (BPH) has not been tested. We compared the miRNome from blood of PCa and BPH patients and further investigated the influence of the tumor volume, tumor-node-metastasis (TNM) classification, Gleason score, pretreatment risk status, and the pretreatment PSA value on the miRNA pattern. By microarray approach, we identified seven miRNAs that were significantly deregulated in PCa patients compared to BPH patients. Using quantitative real time PCR (qRT-PCR), we confirmed downregulation of hsa-miR-221* (now hsa-miR-221-5p) and hsa-miR-708* (now hsa-miR-708-3p) in PCa compared to BPH. Clinical parameters like PSA level, Gleason score, or TNM status seem to have only limited impact on the overall abundance of miRNAs in patients' blood, suggesting a no influence of these factors on the expression of deregulated miRNAs.

[Pharmacological analysis of the effect of natural double-helical nucleic acids on the detoxifying function of the liver].

PubMed

Masycheva, V I; Morozova, E N; Nadolinnaia, I G

1988-10-01

The effect of interferon inductors i.e. double stranded RNAs from S. cerevisiae and phage F6 on the liver detoxicating function was studied on noninbred albino mice. The liver detoxicating function was tested by duration of hexenal sleep. It was shown that intraperitoneal administration of the yeast and phage RNAs in doses of 1/5 LD50 for three times led to increasing of the narcotic sleep duration in the animals by 65 and 207 per cent, respectively. The effect was of the dose-dependent nature. The doses not inducing reliable inhibition of hexenal metabolism were equal to 1/10 LD50 for the yeast dsRNA and 1/27 LD50 for the phage dsRNA. The inhibitory effect of the dsRNAs was retained for 2-3 days after discontinuation of the drug use. When the dsRNAs were administered simultaneously with nembutal, an inductor of the liver microsomal enzymes, the dsRNAs eliminated its inducing effect. Simultaneous administration of alpha-tocopherol lowered the dsRNA effect on hexenal metabolism. The findings suggested that the dsRNA inhibitory effect on the liver detoxicating function was grounded on the mechanisms associated with inhibition of syntheses and activation of lipid peroxidation specific of the monooxygenase system under the action of the dsRNAs.

Smart detection of microRNAs through fluorescence enhancement on a photonic crystal.

PubMed

Pasquardini, L; Potrich, C; Vaghi, V; Lunelli, L; Frascella, F; Descrovi, E; Pirri, C F; Pederzolli, C

2016-04-01

The detection of low abundant biomarkers, such as circulating microRNAs, demands innovative detection methods with increased resolution, sensitivity and specificity. Here, a biofunctional surface was implemented for the selective capture of microRNAs, which were detected through fluorescence enhancement directly on a photonic crystal. To set up the optimal biofunctional surface, epoxy-coated commercially available microscope slides were spotted with specific anti-microRNA probes. The optimal concentration of probe as well as of passivating agent were selected and employed for titrating the microRNA hybridization. Cross-hybridization of different microRNAs was also tested, resulting negligible. Once optimized, the protocol was adapted to the photonic crystal surface, where fluorescent synthetic miR-16 was hybridized and imaged with a dedicated equipment. The photonic crystal consists of a dielectric multilayer patterned with a grating structure. In this way, it is possible to take advantage from both a resonant excitation of fluorophores and an angularly redirection of the emitted radiation. As a result, a significant fluorescence enhancement due to the resonant structure is collected from the patterned photonic crystal with respect to the outer non-structured surface. The dedicated read-out system is compact and based on a wide-field imaging detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development such as for example in microarray-type bioassays. Copyright © 2016 Elsevier B.V. All rights reserved.

Serum miR-206 and other muscle-specific microRNAs as non-invasive biomarkers for Duchenne muscular dystrophy.

PubMed

Hu, Jun; Kong, Min; Ye, Yuanzhen; Hong, Siqi; Cheng, Li; Jiang, Li

2014-06-01

Creatine kinase has been utilized as a diagnostic marker for Duchenne muscular dystrophy (DMD), but it correlates less well with the DMD pathological progression. In this study, we hypothesized that muscle-specific microRNAs (miR-1, -133, and -206) in serum may be useful for monitoring the DMD pathological progression, and explored the possibility of these miRNAs as potential non-invasive biomarkers for the disease. By using real-time quantitative reverse transcription-polymerase chain reaction in a randomized and controlled trial, we detected that miR-1, -133, and -206 were significantly over-expressed in the serum of 39 children with DMD (up to 3.20 ± 1.20, 2(-ΔΔCt) ): almost 2- to 4-fold enriched in comparison to samples from the healthy controls (less than 1.15 ± 0.34, 2(-ΔΔCt) ). To determine whether these miRNAs were related to the clinical features of children with DMD, we analyzed the associations compared to creatine kinase. There were very good inverse correlations between the levels of these miRNAs, especially miR-206, and functional performances: high levels corresponded to low muscle strength, muscle function, and quality of life. Moreover, by receiver operating characteristic curves analyses, we revealed that these miRNAs, especially miR-206, were able to discriminate DMD from controls. Thus, miR-206 and other muscle-specific miRNAs in serum are useful for monitoring the DMD pathological progression, and hence as potential non-invasive biomarkers for the disease. There has been a long-standing need for reliable, non-invasive biomarkers for Duchenne muscular dystrophy (DMD). We found that the levels of muscle-specific microRNAs, especially miR-206, in the serum of DMD were 2- to 4-fold higher than in the controls. High levels corresponded to low muscle strength, muscle function, and quality of life (QoL). These miRNAs were able to discriminate DMD from controls by receiver operating characteristic (ROC) curves analyses. Thus, miR-206 and other muscle-specific miRNAs are useful as non-invasive biomarkers for DMD. © 2014 International Society for Neurochemistry.

Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

PubMed Central

Scott, Jaclyn C.; Brackney, Doug E.; Campbell, Corey L.; Bondu-Hawkins, Virginie; Hjelle, Brian; Ebel, Greg D.; Olson, Ken E.; Blair, Carol D.

2010-01-01

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts. PMID:21049014

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

PubMed Central

Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

2008-01-01

Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms. PMID:18714353

RNA interference technology to control pest sea lampreys--a proof-of-concept.

PubMed

Heath, George; Childs, Darcy; Docker, Margaret F; McCauley, David W; Whyard, Steven

2014-01-01

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

RNA Interference Technology to Control Pest Sea Lampreys - A Proof-of-Concept

PubMed Central

Heath, George; Childs, Darcy; Docker, Margaret F.; McCauley, David W.; Whyard, Steven

2014-01-01

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0–fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species. PMID:24505485

Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells

PubMed Central

Zhao, Yong; Li, Lan; Min, Ling-Jiang; Zhu, Lian-Qin; Sun, Qing-Yuan; Zhang, Hong-Fu; Liu, Xin-Qi; Zhang, Wei-Dong; Ge, Wei; Wang, Jun-Jie; Liu, Jing-Cai

2016-01-01

Zinc oxide (ZnO) nanoparticles (NPs) have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways. PMID:27196542

Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells.

PubMed

Zhao, Yong; Li, Lan; Min, Ling-Jiang; Zhu, Lian-Qin; Sun, Qing-Yuan; Zhang, Hong-Fu; Liu, Xin-Qi; Zhang, Wei-Dong; Ge, Wei; Wang, Jun-Jie; Liu, Jing-Cai; Hao, Zhi-Hui

2016-01-01

Zinc oxide (ZnO) nanoparticles (NPs) have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways.

RNA degradation and models for post-transcriptional gene-silencing.

PubMed

Meins, F

2000-06-01

Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are 'aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.

Genetic Relatedness Among Human Rotaviruses as Determined by RNA Hybridization

PubMed Central

Flores, Jorge; Perez, Irene; White, Laura; Perez, Mireya; Kalica, Anthony R.; Marquina, Ruben; Wyatt, Richard G.; Kapikian, Albert Z.; Chanock, Robert M.

1982-01-01

Viral RNAs from human rotaviruses were compared by gel electrophoresis and by hybridization to probes prepared by in vitro transcription of two well-characterized laboratory strains (Wa and DS-1). Also, the viral RNAs were compared by hybridization to probes prepared from three of the test viruses. Thirteen specimens (diarrheal stools) were obtained from infants and children 5 to 21 months old on a single day at the emergency ward of the Caracas Children's Hospital, and an additional specimen was obtained from the same hospital 6 months before. When the electrophoresed viral RNAs were stained with ethidium bromide and examined by UV light, five different migration patterns (electropherotypes) were distinguished on the basis of differences in mobility of the RNA segments. The hybridization technique that was employed permitted only qualitative comparisons of corresponding genes of different human rotaviruses. Ten of the specimens contained enough virus to yield sufficient RNA for hybridization studies. Eight of the viruses studied by hybridization contained 4 to 11 genes that reacted specifically with the Wa probe to yield double-stranded RNA segments with a mobility similar to that of Wa viral RNA or test virus RNA. The other two viruses contained 11 genes that reacted specifically with the DS-1 hybridization probe to yield double-stranded RNA segments with a mobility similar to DS-1 viral RNA or test virus RNA. A more complex picture emerged when hybridization probes were prepared from three of the test viruses and used to compare the different electropherotypes. Corresponding genes that exhibited similar migration did not necessarily exhibit homology when studied by hybridization. Also, some corresponding genes that exhibited homology did not have the same mobility by gel electrophoresis. Images PMID:6288569

MicroRNA Biomarkers to Generate Sensitivity to Abiraterone-Resistant Prostate Cancer

DTIC Science & Technology

2016-09-01

approach, employing Abiraterone (Abi) plus RNA therapy. For this, we will use an aptamer specific for PSMA (aptPSMA) to specifically target CRPC...develop RNA aptamer therapy. We will test 8 of the recently identified Abi regulated miRNAs for therapeutic utility in vitro. We will design an...as an independent marker for predicting disease relapse. We will use an RNA aptamer which binds specifically to PCa cells to deliver the miRNA. miRNA

A meta-analysis of public microarray data identifies biological regulatory networks in Parkinson's disease.

PubMed

Su, Lining; Wang, Chunjie; Zheng, Chenqing; Wei, Huiping; Song, Xiaoqing

2018-04-13

Parkinson's disease (PD) is a long-term degenerative disease that is caused by environmental and genetic factors. The networks of genes and their regulators that control the progression and development of PD require further elucidation. We examine common differentially expressed genes (DEGs) from several PD blood and substantia nigra (SN) microarray datasets by meta-analysis. Further we screen the PD-specific genes from common DEGs using GCBI. Next, we used a series of bioinformatics software to analyze the miRNAs, lncRNAs and SNPs associated with the common PD-specific genes, and then identify the mTF-miRNA-gene-gTF network. Our results identified 36 common DEGs in PD blood studies and 17 common DEGs in PD SN studies, and five of the genes were previously known to be associated with PD. Further study of the regulatory miRNAs associated with the common PD-specific genes revealed 14 PD-specific miRNAs in our study. Analysis of the mTF-miRNA-gene-gTF network about PD-specific genes revealed two feed-forward loops: one involving the SPRK2 gene, hsa-miR-19a-3p and SPI1, and the second involving the SPRK2 gene, hsa-miR-17-3p and SPI. The long non-coding RNA (lncRNA)-mediated regulatory network identified lncRNAs associated with PD-specific genes and PD-specific miRNAs. Moreover, single nucleotide polymorphism (SNP) analysis of the PD-specific genes identified two significant SNPs, and SNP analysis of the neurodegenerative disease-specific genes identified seven significant SNPs. Most of these SNPs are present in the 3'-untranslated region of genes and are controlled by several miRNAs. Our study identified a total of 53 common DEGs in PD patients compared with healthy controls in blood and brain datasets and five of these genes were previously linked with PD. Regulatory network analysis identified PD-specific miRNAs, associated long non-coding RNA and feed-forward loops, which contribute to our understanding of the mechanisms underlying PD. The SNPs identified in our study can determine whether a genetic variant is associated with PD. Overall, these findings will help guide our study of the complex molecular mechanism of PD.

Comprehensive analysis of a long noncoding RNA-associated competing endogenous RNA network in colorectal cancer.

PubMed

Fan, Qiaowei; Liu, Bingrong

2018-01-01

This study was aimed to develop a lncRNA-associated competing endogenous RNA (ceRNA) network to provide further understanding of the ceRNA regulatory mechanism and pathogenesis in colorectal cancer (CRC). Expression profiles of mRNAs, lncRNAs, and miRNAs, and clinical information for CRC patients were obtained from The Cancer Genome Atlas. The differentially expressed mRNAs, lncRNAs, and miRNAs (referred to as "DEmRNAs", "DElncRNAs", and "DEmiRNAs", respectively) were screened out between 539 CRC samples and 11 normal samples. The interactions between DElncRNAs and DEmiRNAs were predicted by miRcode. The DEmRNAs targeted by the DEmiRNAs were retrieved according to TargetScan, miRTar-Base, and miRDB. The lncRNA-miRNA-mRNA ceRNA network was constructed based on the DEmiRNA-DElncRNA and DEmiRNA-DEmRNA interactions. Functional enrichment analysis revealed the biological processes and pathways of DEmRNAs involved in the development of CRC. Key lncRNAs were further analyzed for their associations with overall survival and clinical features of CRC patients. A total of 1,767 DEmRNAs, 608 DElncRNAs, and 283 DEmiRNAs were identified as CRC-specific RNAs. Three hundred eighty-two DEmiRNA-DElncRNA interactions and 68 DEmiRNA-DEmRNA interactions were recognized according to the relevant databases. The lncRNA-miRNA-mRNA ceRNA network was constructed using 25 DEmiRNAs, 52 DEmRNAs, and 64 DElncRNAs. Two DElncRNAs, five DEmiRNAs, and six DEmRNAs were demonstrated to be related to the prognosis of CRC patients. Four DElncRNAs were found to be associated with clinical features. Twenty-eight Gene Ontology terms and 10 Kyoto Encyclopedia of Genes and Genomes pathways were found to be significantly enriched by the DEmRNAs in the ceRNA network. Our results showed cancer-specific mRNA, lncRNA, and miRNA expression patterns and enabled us to construct an lncRNA-associated ceRNA network that provided new insights into the molecular mechanisms of CRC. Key RNA transcripts related to the overall survival and clinical features were also found with promising potential as biomarkers for diagnosis, survival prediction, and classification of CRC.

Intramyocardial Injection of siRNAs Can Efficiently Establish Myocardial Tissue-Specific Renalase Knockdown Mouse Model.

PubMed

Huang, Kun; Liu, Ju; Zhang, Hui; Wang, Jiliang; Li, Huili

2016-01-01

Ischaemia/reperfusion (I/R) injury will cause additional death of cardiomyocytes in ischaemic heart disease. Recent studies revealed that renalase was involved in the I/R injury. So, the myocardial tissue-specific knockdown mouse models were needed for the investigations of renalase. To establish the mouse models, intramyocardial injection of siRNAs targeting renalase was performed in mice. The wild distribution and high transfection efficiency of the siRNAs were approved. And the renalase expression was efficiently suppressed in myocardial tissue. Compared with the high cost, time consumption, and genetic compensation risk of the Cre/loxP technology, RNA interference (RNAi) technology is much cheaper and less time-consuming. Among the RNAi technologies, injection of siRNAs is safer than virus. And considering the properties of the I/R injury mouse models, the efficiency and durability of injection with siRNAs are acceptable for the studies. Altogether, intramyocardial injection of siRNAs targeting renalase is an economical, safe, and efficient method to establish myocardial tissue-specific renalase knockdown mouse models.

Noncoding RNAs and immune checkpoints-clinical implications as cancer therapeutics.

PubMed

Smolle, Maria A; Calin, Horatiu N; Pichler, Martin; Calin, George A

2017-07-01

A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice. © 2017 Federation of European Biochemical Societies.

Implication of microRNAs in drug resistance for designing novel cancer therapy

PubMed Central

Sarkar, Fazlul H; Li, Yiwei; Wang, Zhiwei; Kong, Dejuan; Ali, Shadan

2010-01-01

Recently, microRNAs (miRNAs) have received increasing attention in the field of cancer research. miRNAs play important roles in many normal biological processes; however, the aberrant miRNA expression and its correlation with the development and progression of cancers is an emerging field. Therefore, miRNAs could be used as biomarkers for diagnosis of cancer and prediction of prognosis. Importantly, some miRNAs could regulate the formation of cancer stem cells and the acquisition of epithelial-mesenchymal transition, which are critically associated with drug resistance. Moreover, some miRNAs could target genes related to drug-sensitivity, resulting in the altered sensitivity of cancer cells to anti-cancer drugs. Emerging evidences have also shown that knock-down or re-expression of specific miRNAs by synthetic antisense oligonucleotides or pre-miRNAs could induce drug sensitivity, leading to increased inhibition of cancer cell growth, invasion, and metastasis. More importantly, recent studies have shown that natural agents including isoflavone, 3,3′-diindolylmethane, and (−)-epigallocatechin-3-gallate altered miRNA expression profiles, leading to an increased sensitivity of cancer cells to conventional therapeutics. These emerging results suggest that specific targeting of miRNAs by different approaches could open new avenues for cancer treatment through overcoming drug resistance and thereby improve the outcome of cancer therapy. PMID:20236855

Urine podocyte mRNAs mark disease activity in IgA nephropathy

PubMed Central

Fukuda, Akihiro; Sato, Yuji; Iwakiri, Takashi; Komatsu, Hiroyuki; Kikuchi, Masao; Kitamura, Kazuo; Wiggins, Roger C.; Fujimoto, Shouichi

2015-01-01

Background Podocyte depletion is a major mechanism driving glomerulosclerosis. We and others have previously projected from model systems that podocyte-specific mRNAs in the urine pellet might serve as glomerular disease markers. We evaluated IgA nephropathy (IgAN) to test this concept. Methods From 2009 to 2013, early morning voided urine samples and kidney biopsies from IgAN patients (n = 67) were evaluated in comparison with urine samples from healthy age-matched volunteers (n = 28). Urine podocyte (podocin) mRNA expressed in relation to either urine creatinine concentration or a kidney tubular marker (aquaporin 2) was tested as markers. Results Urine podocyte mRNAs were correlated with the severity of active glomerular lesions (segmental glomerulosclerosis and acute extracapillary proliferation), but not with non-glomerular lesions (tubular atrophy/interstitial fibrosis) or with clinical parameters of kidney injury (serum creatinine and estimated glomerular filtration rate), or with degree of accumulated podocyte loss at the time of biopsy. In contrast, proteinuria correlated with all histological and clinical markers. Glomerular tuft podocyte nuclear density (a measure of cumulative podocyte loss) correlated with tubular atrophy/interstitial fibrosis, estimated-glomerular filtration rate and proteinuria, but not with urine podocyte markers. In a subset of the IgA cohort (n = 19, median follow-up period = 37 months), urine podocyte mRNAs were significantly decreased after treatment, in contrast to proteinuria which was not significantly changed. Conclusions Urine podocyte mRNAs reflect active glomerular injury at a given point in time, and therefore provide both different and additional clinical information that can complement proteinuria in the IgAN decision-making paradigm. PMID:25956757

Nephron segment specific microRNA biomarkers of pre-clinical drug-induced renal toxicity: Opportunities and challenges

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nassirpour, Rounak, E-mail: Rounak.nassirpour@pfiz

Drug-induced nephrotoxicity is a common drug development complication for pharmaceutical companies. Sensitive, specific, translatable and non-invasive biomarkers of renal toxicity are urgently needed to diagnose nephron segment specific injury. The currently available gold standard biomarkers for nephrotoxicity are not kidney-specific, lack sensitivity for early detection, and are not suitable for renal damage localization (glomerular vs tubulointerstitial injury). MicroRNAs (miRNAs) are increasingly gaining momentum as promising biomarkers of various organ toxicities, including drug induced renal injury. This is mostly due to their stability in easily accessible biofluids, ease of developing nucleic acids detection compared to protein detection assays, as well asmore » their interspecies translatability. Increasing concordance of miRNA findings by standardizing methodology most suitable for their detection and quantitation, as well as characterization of their expression pattern in a cell type specific manner, will accelerate progress toward validation of these miRNAs as biomarkers in pre-clinical, and clinical settings. This review aims to highlight the current pre-clinical findings surrounding miRNAs as biomarkers in two important segments of the nephron, the glomerulus and tubules. - Highlights: • miRNAs are promising biomarkers of drug-induced kidney injury. • Summarized pre-clinical miRNA biomarkers of drug-induced nephrotoxicity. • Described the strengths and challenges associated with miRNAs as biomarkers.« less

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Cross-species inference of long non-coding RNAs greatly expands the ruminant transcriptome.

PubMed

Bush, Stephen J; Muriuki, Charity; McCulloch, Mary E B; Farquhar, Iseabail L; Clark, Emily L; Hume, David A

2018-04-24

mRNA-like long non-coding RNAs (lncRNAs) are a significant component of mammalian transcriptomes, although most are expressed only at low levels, with high tissue-specificity and/or at specific developmental stages. Thus, in many cases lncRNA detection by RNA-sequencing (RNA-seq) is compromised by stochastic sampling. To account for this and create a catalogue of ruminant lncRNAs, we compared de novo assembled lncRNAs derived from large RNA-seq datasets in transcriptional atlas projects for sheep and goats with previous lncRNAs assembled in cattle and human. We then combined the novel lncRNAs with the sheep transcriptional atlas to identify co-regulated sets of protein-coding and non-coding loci. Few lncRNAs could be reproducibly assembled from a single dataset, even with deep sequencing of the same tissues from multiple animals. Furthermore, there was little sequence overlap between lncRNAs that were assembled from pooled RNA-seq data. We combined positional conservation (synteny) with cross-species mapping of candidate lncRNAs to identify a consensus set of ruminant lncRNAs and then used the RNA-seq data to demonstrate detectable and reproducible expression in each species. In sheep, 20 to 30% of lncRNAs were located close to protein-coding genes with which they are strongly co-expressed, which is consistent with the evolutionary origin of some ncRNAs in enhancer sequences. Nevertheless, most of the lncRNAs are not co-expressed with neighbouring protein-coding genes. Alongside substantially expanding the ruminant lncRNA repertoire, the outcomes of our analysis demonstrate that stochastic sampling can be partly overcome by combining RNA-seq datasets from related species. This has practical implications for the future discovery of lncRNAs in other species.

Genome-wide characterization of microRNA in foxtail millet (Setaria italica)

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood. Results In this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing. Conclusions We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally. PMID:24330712

Genome-wide characterization of microRNA in foxtail millet (Setaria italica).

PubMed

Yi, Fei; Xie, Shaojun; Liu, Yuwei; Qi, Xin; Yu, Jingjuan

2013-12-13

MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood. In this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing. We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally.

Endothelial and circulating C19MC microRNAs are biomarkers of infantile hemangioma

PubMed Central

Strub, Graham M.; Kirsh, Andrew L.; Whipple, Mark E.; Kuo, Winston P.; Keller, Rachel B.; Kapur, Raj P.; Majesky, Mark W.; Perkins, Jonathan A.

2016-01-01

Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1–expressing (GLUT-1–expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response. PMID:27660822

Posttranscriptional regulation of lipid metabolism by non-coding RNAs and RNA binding proteins.

PubMed

Singh, Abhishek K; Aryal, Binod; Zhang, Xinbo; Fan, Yuhua; Price, Nathan L; Suárez, Yajaira; Fernández-Hernando, Carlos

2017-11-29

Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of MicroRNAs-Mediated Autophagic Flux: A New Regulatory Avenue for Neurodegenerative Diseases With Focus on Prion Diseases

PubMed Central

Shah, Syed Zahid Ali; Zhao, Deming; Hussain, Tariq; Sabir, Naveed; Yang, Lifeng

2018-01-01

Prion diseases are fatal neurological disorders affecting various mammalian species including humans. Lack of proper diagnostic tools and non-availability of therapeutic remedies are hindering the control strategies for prion diseases. MicroRNAs (miRNAs) are abundant endogenous short non-coding essential RNA molecules that negatively regulate the target genes after transcription. Several biological processes depend on miRNAs, and altered profiles of these miRNAs are potential biomarkers for various neurodegenerative diseases, including prion diseases. Autophagic flux degrades the misfolded prion proteins to reduce chronic endoplasmic reticulum stress and enhance cell survival. Recent evidence suggests that specific miRNAs target and regulate the autophagic mechanism, which is critical for alleviating cellular stress. miRNAs-mediated regulation of these specific proteins involved in the autophagy represents a new target with highly significant therapeutic prospects. Here, we will briefly describe the biology of miRNAs, the use of miRNAs as potential biomarkers with their credibility, the regulatory mechanism of miRNAs in major neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and prion diseases, degradation pathways for aggregated prion proteins, the role of autophagy in prion diseases. Finally, we will discuss the miRNAs-modulated autophagic flux in neurodegenerative diseases and employ them as potential therapeutic intervention strategy in prion diseases. PMID:29867448

High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

PubMed Central

Islam, Md. Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

2015-01-01

MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops. PMID:25861616

Circular RNA expression profiles and features in human tissues: a study using RNA-seq data.

PubMed

Xu, Tianyi; Wu, Jing; Han, Ping; Zhao, Zhongming; Song, Xiaofeng

2017-10-03

Circular RNA (circRNA) is one type of noncoding RNA that forms a covalently closed continuous loop. Similar to long noncoding RNA (lncRNA), circRNA can act as microRNA (miRNA) 'sponges' to regulate gene expression, and its abnormal expression is related to diseases such as atherosclerosis, nervous system disorders and cancer. So far, there have been no systematic studies on circRNA abundance and expression profiles in human adult and fetal tissues. We explored circRNA expression profiles using RNA-seq data for six adult and fetal normal tissues (colon, heart, kidney, liver, lung, and stomach) and four gland normal tissues (adrenal gland, mammary gland, pancreas, and thyroid gland). A total of 8120, 25,933 and 14,433 circRNAs were detected by at least two supporting junction reads in adult, fetal and gland tissues, respectively. Among them, 3092, 14,241 and 6879 circRNAs were novel when compared to the published results. In each adult tissue type, we found at least 1000 circRNAs, among which 36.97-50.04% were tissue-specific. We reported 33 circRNAs that were ubiquitously expressed in all the adult tissues we examined. To further explore the potential "housekeeping" function of these circRNAs, we constructed a circRNA-miRNA-mRNA regulatory network containing 17 circRNAs, 22 miRNAs and 90 mRNAs. Furthermore, we found that both the abundance and the relative expression level of circRNAs were higher in fetal tissue than adult tissue. The number of circRNAs in gland tissues, especially in mammary gland (9665 circRNA candidates), was higher than that of other adult tissues (1160-3777). We systematically investigated circRNA expression in a variety of human adult and fetal tissues. Our observation of different expression level of circRNAs in adult and fetal tissues suggested that circRNAs might play their role in a tissue-specific and development-specific fashion. Analysis of circRNA-miRNA-mRNA network provided potential targets of circRNAs. High expression level of circRNAs in mammary gland might be attributed to the rich innervation.

piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism.

PubMed

Beyret, Ergin; Liu, Na; Lin, Haifan

2012-10-01

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5' Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.

An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes

PubMed Central

Li, Xin Zhiguo; Roy, Christian K.; Dong, Xianjun; Bolcun-Filas, Ewelina; Wang, Jie; Han, Bo W.; Xu, Jia; Moore, Melissa J.; Schimenti, John C.; Weng, Zhiping; Zamore, Phillip D.

2013-01-01

SUMMARY Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PMID:23523368

Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases.

PubMed

Gstir, Ronald; Schafferer, Simon; Scheideler, Marcel; Misslinger, Matthias; Griehl, Matthias; Daschil, Nina; Humpel, Christian; Obermair, Gerald J; Schmuckermair, Claudia; Striessnig, Joerg; Flucher, Bernhard E; Hüttenhofer, Alexander

2014-12-01

We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson's disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer's disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer's disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs. © 2014 Gstir et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Role of RNA interference (RNAi) in the Moss Physcomitrella patens.

PubMed

Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel

2013-01-14

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species.

Long Distance Metabolic Regulation through Adipose-Derived Circulating Exosomal miRNAs: A Trail for RNA-Based Therapies?

PubMed Central

Fatima, Farah; Nawaz, Muhammad

2017-01-01

The contribution of non-coding RNAs, such as microRNAs (miRNAs) in regulating physiological and pathological states has been intensively elucidated during last 15 years. The discovery of circulating miRNAs (cir-miRNAs) in variety of body fluids, is, however a recent focus of interest in understanding pathophysiological states of their originating cells/organs. Yet another stimulating debate that takes miRNAs to the next level is their presence in exosomes, and this is truly interesting area of research. Exosomes are cell-derived extracellular vesicles, and are naturally equipped biological vehicles that not only enable functional transfer of miRNAs between cells (horizontal transfer) but also foster inter-organ communication, presumably guided by organ specific receptors—decorated on their surface. However, understandings on inter-organ communication elicited by tissue specific exosomal-miRNA fingerprints remain elusive. Recently, Thomou et al., has discovered that adipose tissue contributes a large fraction of adipose specific exosomal-miRNA fingerprints in blood circulation. Experimental evidence emphasize adipose tissue as major depot of cir-miRNAs that sail through blood flow and reach to distal organs—primarily in the liver, where they regulate gene expression of host tissue and elicit metabolic control. This appears to be a genetic form of adipokines (endocrine factors secreted from adipose tissue). We review such offshore metabolic insults, and make an effort to address few important missing links between miRNAs processing and their incorporation into exosomes. We provide potential perspectives on how this knowledge could be steered towards RNA-based therapeutics for monitoring complex metabolic diseases and beyond. PMID:28824444

The microRNA-processing enzyme Dicer is essential for thyroid function.

PubMed

Frezzetti, Daniela; Reale, Carla; Calì, Gaetano; Nitsch, Lucio; Fagman, Henrik; Nilsson, Ola; Scarfò, Marzia; De Vita, Gabriella; Di Lauro, Roberto

2011-01-01

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.

Genome-wide Identification and characterization of circular RNAs in the rice blast fungus Magnaporthe oryzae.

PubMed

Yuan, Jialan; Wang, Zhao; Xing, Junjie; Yang, Qingyong; Chen, Xiao-Lin

2018-04-30

Numerous circRNAs have been identified in different organisms, but little attention has been addressed on fungal circRNAs. Here, we identified a total of 8,848 circRNAs from the model plant pathogenic fungus M. oryzae. 5,840 circRNAs were identified from mycelium, 2,721 circRNAs from conidium, while only 287 circRNAs from both tissues. This indicated that most of the M. oryzae circRNAs were specifically expressed in mycelium or in conidium. Parental genes of circRNAs in mycelium were enriched in basic metabolisms required for normal growth, while in conidium, they were enriched in biogenesis of storages potentially used for infection. M. oryzae circRNAs could also bind to miRNAs, suggesting they may also function as sponges in fungi. This study suggested M. oryzae circRNAs could play important roles in regulation of growth and development.

Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

PubMed

Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

2010-04-01

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

Long non-coding RNAs are associated with spatiotemporal gene expression profiles in the marine gastropod Tegula atra.

PubMed

Détrée, Camille; Núñez-Acuña, Gustavo; Tapia, Fabian; Gallardo-Escárate, Cristian

2017-06-01

Increasing evidence suggests that long non-coding RNAs (lncRNAs) play diverse roles in cellular processes, including in the regulation of embryogenesis and growth. However, little is known about the role of lncRNAs in marine invertebrates inhabiting changing environments. Therefore, the aim of this study was to present the first characterization of lncRNAs in an intertidal marine gastropod. Specifically, Tegula atra individuals were sampled in four sites of the central-northern Chilean coastline (28-31°) during summer and winter. A pipeline was constructed, and 3524 putative lncRNAs were identified from transcriptome databases specific to T. atra. These lncRNAs exhibited characteristics common to known lncRNAs, including a length shorter than coding sequences, low GC-content, and low sequence conservation. Expression analyses revealed that lncRNAs varied more in the summer. Furthermore, a majority of the differentially expressed lncRNAs were found in the southernmost population, the seasonal temperatures of which varied the greatest among all groups. Additionally, co-expression analysis found some lncRNAs strongly correlated with coding genes involved in the environmental stress response, such as heat shock proteins and metalloproteins. In contrast, other lncRNA expressions were strongly uncorrelated with genes involved in lipid/carbohydrates metabolism and cell-cell communication. This study provides the first large-scale characterization of lncRNAs in a marine gastropod, with results suggesting a putative role of lncRNAs in thermal tolerance, as well as an association with molecular mechanisms involved in the local adaptations of marine invertebrate populations. Copyright © 2017 Elsevier B.V. All rights reserved.

The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets

PubMed Central

Vakiloroayaei, Ana; Shah, Neha S.; Oeffinger, Marlene

2017-01-01

Abstract Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3′OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates. PMID:28977649

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

PubMed

Xu, Li; Zhao, Lixia; Gao, Yandi; Xu, Jing; Han, Renzhi

2017-03-17

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Highly Predictive Model for Diagnosis of Colorectal Neoplasms Using Plasma MicroRNA: Improving Specificity and Sensitivity

PubMed Central

Carter, Jane V.; Roberts, Henry L.; Pan, Jianmin; Rice, Jonathan D.; Burton, James F.; Galbraith, Norman J.; Eichenberger, M. Robert; Jorden, Jeffery; Deveaux, Peter; Farmer, Russell; Williford, Anna; Kanaan, Ziad; Rai, Shesh N.; Galandiuk, Susan

2016-01-01

OBJECTIVE(S) Develop a plasma-based microRNA (miRNA) diagnostic assay specific for colorectal neoplasms, building upon our prior work. BACKGROUND Colorectal neoplasms (colorectal cancer [CRC] and colorectal advanced adenoma [CAA]) frequently develop in individuals at ages when other common cancers also occur. Current screening methods lack sensitivity, specificity, and have poor patient compliance. METHODS Plasma was screened for 380 miRNAs using microfluidic array technology from a “Training” cohort of 60 patients, (10 each) control, CRC, CAA, breast (BC), pancreatic (PC) and lung (LC) cancer. We identified uniquely dysregulated miRNAs specific for colorectal neoplasia (p<0.05, false discovery rate: 5%, adjusted α=0.0038). These miRNAs were evaluated using single assays in a “Test” cohort of 120 patients. A mathematical model was developed to predict blinded sample identity in a 150 patient “Validation” cohort using repeat-sub-sampling validation of the testing dataset with 1000 iterations each to assess model detection accuracy. RESULTS Seven miRNAs (miR-21, miR-29c, miR-122, miR-192, miR-346, miR-372, miR-374a) were selected based upon p-value, area-under-the-curve (AUC), fold-change, and biological plausibility. AUC (±95% CI) for “Test” cohort comparisons were 0.91 (0.85-0.96), 0.79 (0.70-0.88) and 0.98 (0.96-1.0), respectively. Our mathematical model predicted blinded sample identity with 69-77% accuracy between all neoplasia and controls, 67-76% accuracy between colorectal neoplasia and other cancers, and 86-90% accuracy between colorectal cancer and colorectal adenoma. CONCLUSIONS Our plasma miRNA assay and prediction model differentiates colorectal neoplasia from patients with other neoplasms and from controls with higher sensitivity and specificity compared to current clinical standards. PMID:27471839

Re-analysis of long non-coding RNAs and prediction of circRNAs reveal their novel roles in susceptible tomato following TYLCV infection.

PubMed

Wang, Jinyan; Yang, Yuwen; Jin, Lamei; Ling, Xitie; Liu, Tingli; Chen, Tianzi; Ji, Yinghua; Yu, Wengui; Zhang, Baolong

2018-06-04

Long Noncoding-RNAs (LncRNAs) are known to be involved in some biological processes, but their roles in plant-virus interactions remain largely unexplored. While circular RNAs (circRNAs) have been studied in animals, there has yet to be extensive research on them in a plant system, especially in tomato-tomato yellow leaf curl virus (TYLCV) interaction. In this study, RNA transcripts from the susceptible tomato line JS-CT-9210 either infected with TYLCV or untreated, were sequenced in a pair-end strand-specific manner using ribo-zero rRNA removal library method. A total of 2056 lncRNAs including 1767 long intergenic non-coding RNA (lincRNAs) and 289 long non-coding natural antisense transcripts (lncNATs) were obtained. The expression patterns in lncRNAs were similar in susceptible tomato plants between control check (CK) and TYLCV infected samples. Our analysis suggested that lncRNAs likely played a role in a variety of functions, including plant hormone signaling, protein processing in the endoplasmic reticulum, RNA transport, ribosome function, photosynthesis, glulathione metabolism, and plant-pathogen interactions. Using virus-induced gene silencing (VIGS) analysis, we found that reduced expression of the lncRNA S-slylnc0957 resulted in enhanced resistance to TYLCV in susceptible tomato plants. Moreover, we identified 184 circRNAs candidates using the CircRNA Identifier (CIRI) software, of which 32 circRNAs were specifically expressed in untreated samples and 83 circRNAs in TYLCV samples. Approximately 62% of these circRNAs were derived from exons. We validated the circRNAs by both PCR and Sanger sequencing using divergent primers, and found that most of circRNAs were derived from the exons of protein coding genes. The silencing of these circRNAs parent genes resulted in decreased TYLCV virus accumulation. In this study, we identified novel lncRNAs and circRNAs using bioinformatic approaches and showed that these RNAs function as negative regulators of TYLCV infection. Moreover, the expression patterns of lncRNAs in susceptible tomato plants were different from that of resistant tomato plants, while exonic circRNAs expression positively associated with their respective protein coding genes. This work provides a foundation for elaborating the novel roles of lncRNAs and circRNAs in susceptible tomatoes following TYLCV infection.

Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance.

PubMed

Leidinger, Petra; Backes, Christina; Rheinheimer, Stefanie; Keller, Andreas; Meese, Eckart

2015-01-01

Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays. While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding. The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions.

MicroRNA-1 and -133 Increase Arrhythmogenesis in Heart Failure by Dissociating Phosphatase Activity from RyR2 Complex

PubMed Central

Belevych, Andriy E.; Sansom, Sarah E.; Terentyeva, Radmila; Ho, Hsiang-Ting; Nishijima, Yoshinori; Martin, Mickey M.; Jindal, Hitesh K.; Rochira, Jennifer A.; Kunitomo, Yukiko; Abdellatif, Maha; Carnes, Cynthia A.; Elton, Terry S.; Györke, Sandor; Terentyev, Dmitry

2011-01-01

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis. PMID:22163007

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

PubMed

Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2017-10-01

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

PubMed Central

2012-01-01

Background In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet. Results Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries. Conclusions Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype. PMID:22452797

CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

PubMed

van Wolfswinkel, Josien C; Claycomb, Julie M; Batista, Pedro J; Mello, Craig C; Berezikov, Eugene; Ketting, René F

2009-10-02

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

PubMed

Yamasaki, Tomohito; Voshall, Adam; Kim, Eun-Jeong; Moriyama, Etsuko; Cerutti, Heriberto; Ohama, Takeshi

2013-12-01

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers.

PubMed

Yun, Seok Joong; Jeong, Pildu; Kang, Ho Won; Kim, Ye-Hwan; Kim, Eun-Ah; Yan, Chunri; Choi, Young-Ki; Kim, Dongho; Kim, Jung Min; Kim, Seon-Kyu; Kim, Seon-Young; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Koh, Gou-Young; Moon, Sung-Kwon; Kim, Isaac Yi; Kim, Jayoung; Choi, Yung-Hyun; Kim, Wun-Jae

2015-06-01

MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone.

Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers

PubMed Central

Yun, Seok Joong; Jeong, Pildu; Kang, Ho Won; Kim, Ye-Hwan; Kim, Eun-Ah; Yan, Chunri; Choi, Young-Ki; Kim, Dongho; Kim, Jung Min; Kim, Seon-Kyu; Kim, Seon-Young; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Koh, Gou-Young; Moon, Sung-Kwon; Kim, Isaac Yi; Kim, Jayoung; Choi, Yung-Hyun; Kim, Wun-Jae

2015-01-01

Purpose: MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. Methods: In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. Results: The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. Conclusions: Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone. PMID:26126436

Altered Viral Replication and Cell Responses by Inserting MicroRNA Recognition Element into PB1 in Pandemic Influenza A Virus (H1N1) 2009

PubMed Central

Shen, Xiaoyue; Sun, Wenkui; Shi, Yi; Xing, Zheng; Su, Xin

2015-01-01

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. PMID:25788763

RNA binding specificity of Ebola virus transcription factor VP30.

PubMed

Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

2016-09-01

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

Cerebellar neurodegeneration in the absence of microRNAs

PubMed Central

Schaefer, Anne; O'Carroll, Dónal; Tan, Chan Lek; Hillman, Dean; Sugimori, Mutsuyuki; Llinas, Rodolfo; Greengard, Paul

2007-01-01

Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders. PMID:17606634

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

PubMed

Roy, Sribash; Tripathi, Abhinandan Mani; Yadav, Amrita; Mishra, Parneeta; Nautiyal, Chandra Shekhar

2016-01-01

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

Regulation of microRNAs in Cancer Metastasis

PubMed Central

Bouyssou, Juliette M.C.; Manier, Salomon; Huynh, Daisy; Issa, Samar; Roccaro, Aldo M.; Ghobrial, Irene M.

2014-01-01

Metastasis is a phenomenon of crucial importance in defining prognosis in patients with cancer and is often responsible for cancer-related mortality. It is known that several steps are necessary for clonal cells to disseminate from their primary tumor site and colonize distant tissues, thus originating metastatic lesions. Therefore, investigating the molecular actors regulating this process may provide helpful insights in the development of efficient therapeutic responses. Recent evidences have indicated the role of microRNAs (miRNAs) in modulating the metastatic process in solid tumors. miRNAs are small regulatory non-coding RNAs that bind specific target mRNAs, leading to translational repression. miRNAs are known to act as negative regulators of gene expression and are involved in the regulation of biological processes, including cell growth, differentiation and apoptosis, both in physiological conditions and during diseases, such as tumors. In the specific field of tumorigenesis, miRNAs play an important role in mediating oncogenesis and favoring tumor progression, as a result of their ability to modulate epithelial-to-mesenchymal transition (EMT) and other series of events facilitating the formation of metastasis. The role of miRNAs in cancer development has been widely studied and has helped elucidate events such as the change in expression of oncogenes, tumor-suppressors and cancer-related proteins. This review focuses on the mechanisms underlying the role of miRNAs as part of the metastatic process. PMID:24569228

MicroRNAs as targets for dietary and pharmacological inhibitors of mutagenesis and carcinogenesis

PubMed Central

Izzotti, Alberto; Cartiglia, Cristina; Steele, Vernon E.; De Flora, Silvio

2012-01-01

MicroRNAs (miRNAs) have been implicated in many biological processes, cancer, and other diseases. In addition, miRNAs are dysregulated following exposure to toxic and genotoxic agents. Here we review studies evaluating modulation of miRNAs by dietary and pharmacological agents, which could potentially be exploited for inhibition of mutagenesis and carcinogenesis. This review covers natural agents, including vitamins, oligoelements, polyphenols, isoflavones, indoles, isothiocyanates, phospholipids, saponins, anthraquinones and polyunsaturated fatty acids, and synthetic agents, including thiols, nuclear receptor agonists, histone deacetylase inhibitors, antiinflammatory drugs, and selective estrogen receptor modulators. As many as 145 miRNAs, involved in the control of a variety of carcinogenesis mechanisms, were modulated by these agents, either individually or in combination. Most studies used cancer cells in vitro with the goal of modifying their phenotype by changing miRNA expression profiles. In vivo studies evaluated regulation of miRNAs by chemopreventive agents in organs of mice and rats, either untreated or exposed to carcinogens, with the objective of evaluating their safety and efficacy. The tissue specificity of miRNAs could be exploited for the chemoprevention of site-specific cancers, and the study of polymorphic miRNAs is expected to predict the individual response to chemopreventive agents as a tool for developing new prevention strategies. PMID:22683846

A Diversity of Conserved and Novel Ovarian MicroRNAs in the Speckled Wood (Pararge aegeria)

PubMed Central

Quah, Shan; Breuker, Casper J.; Holland, Peter W. H.

2015-01-01

microRNAs (miRNAs) are important regulators of animal development and other processes, and impart robustness to living systems through post-transcriptional regulation of specific mRNA transcripts. It is postulated that newly emergent miRNAs are generally expressed at low levels and with spatiotemporally restricted expression domains, thus minimising effects of spurious targeting on animal transcriptomes. Here we present ovarian miRNA transcriptome data for two geographically distinct populations of the Speckled Wood butterfly (Pararge aegeria). A total of 74 miRNAs were identified, including 11 newly discovered and evolutionarily-young miRNAs, bringing the total of miRNA genes known from P. aegeria up to 150. We find a positive correlation between miRNA age and expression level. A common set of 55 miRNAs are expressed in both populations. From this set, we identify seven that are consistently either ovary-specific or highly upregulated in ovaries relative to other tissues. This ‘ovary set’ includes miRNAs with known contributions to ovarian function in other insect species with similar ovaries and mode of oogenesis, including miR-989 and miR-2763, plus new candidates for ovarian function. We also note that conserved miRNAs are overrepresented in the ovary relative to the whole body. PMID:26556800

MicroRNA screening identifies a link between NOVA1 expression and a low level of IKAP in familial dysautonomia

PubMed Central

Hervé, Mylène

2016-01-01

ABSTRACT Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a mutation in intron 20 of the IKBKAP gene (c.2204+6T>C), leading to tissue-specific skipping of exon 20 and a decrease in the synthesis of the encoded protein IKAP (also known as ELP1). Small non-coding RNAs known as microRNAs (miRNAs) are important post-transcriptional regulators of gene expression and play an essential role in the nervous system development and function. To better understand the neuronal specificity of IKAP loss, we examined expression of miRNAs in human olfactory ecto-mesenchymal stem cells (hOE-MSCs) from five control individuals and five FD patients. We profiled the expression of 373 miRNAs using microfluidics and reverse transcription coupled to quantitative PCR (RT-qPCR) on two biological replicate series of hOE-MSC cultures from healthy controls and FD patients. This led to the total identification of 26 dysregulated miRNAs in FD, validating the existence of a miRNA signature in FD. We then selected the nine most discriminant miRNAs for further analysis. The signaling pathways affected by these dysregulated miRNAs were largely within the nervous system. In addition, many targets of these dysregulated miRNAs had been previously demonstrated to be affected in FD models. Moreover, we found that four of our nine candidate miRNAs target the neuron-specific splicing factor NOVA1. We demonstrated that overexpression of miR-203a-3p leads to a decrease of NOVA1, counter-balanced by an increase of IKAP, supporting a potential interaction between NOVA1 and IKAP. Taken together, these results reinforce the choice of miRNAs as potential therapeutic targets and suggest that NOVA1 could be a regulator of FD pathophysiology. PMID:27483351

[Long non-coding RNAs in the pathophysiology of atherosclerosis].

PubMed

Novak, Jan; Vašků, Julie Bienertová; Souček, Miroslav

2018-01-01

The human genome contains about 22 000 protein-coding genes that are transcribed to an even larger amount of messenger RNAs (mRNA). Interestingly, the results of the project ENCODE from 2012 show, that despite up to 90 % of our genome being actively transcribed, protein-coding mRNAs make up only 2-3 % of the total amount of the transcribed RNA. The rest of RNA transcripts is not translated to proteins and that is why they are referred to as "non-coding RNAs". Earlier the non-coding RNA was considered "the dark matter of genome", or "the junk", whose genes has accumulated in our DNA during the course of evolution. Today we already know that non-coding RNAs fulfil a variety of regulatory functions in our body - they intervene into epigenetic processes from chromatin remodelling to histone methylation, or into the transcription process itself, or even post-transcription processes. Long non-coding RNAs (lncRNA) are one of the classes of non-coding RNAs that have more than 200 nucleotides in length (non-coding RNAs with less than 200 nucleotides in length are called small non-coding RNAs). lncRNAs represent a widely varied and large group of molecules with diverse regulatory functions. We can identify them in all thinkable cell types or tissues, or even in an extracellular space, which includes blood, specifically plasma. Their levels change during the course of organogenesis, they are specific to different tissues and their changes also occur along with the development of different illnesses, including atherosclerosis. This review article aims to present lncRNAs problematics in general and then focuses on some of their specific representatives in relation to the process of atherosclerosis (i.e. we describe lncRNA involvement in the biology of endothelial cells, vascular smooth muscle cells or immune cells), and we further describe possible clinical potential of lncRNA, whether in diagnostics or therapy of atherosclerosis and its clinical manifestations.Key words: atherosclerosis - lincRNA - lncRNA - MALAT - MIAT.

MicroRNAs and cancer: a meeting summary of the eponymous Keystone Conference.

PubMed

Godshalk, Sirie E; Melnik-Martinez, Katya V; Pasquinelli, Amy E; Slack, Frank J

2010-02-16

This report summarizes the information presented at the 2009 Keystone Conference on MicroRNAs and Cancer, held in Keystone, Colorado, USA, June 10th to 15th 2009. Soon after microRNAs (miRNAs) emerged as an abundant new class of non-coding RNAs (ncRNAs), evidence started to mount supporting important roles for these regulatory RNAs in human health and disease. Mis-regulation of specific miRNA pathways has been linked to diverse cancers. The recent Keystone meeting highlighted progress in understanding the role of miRNAs in normal development and oncogenesis. Recurring themes included the complexities associated with miRNA biogenesis, target recognition, elucidation of genetic networks where miRNAs play pivotal roles often within feedback loops, and the promise of small RNAs as diagnostics and therapeutics in combating cancer.

Circular RNAs: Regulators of Cancer-Related Signaling Pathways and Potential Diagnostic Biomarkers for Human Cancers

PubMed Central

Yang, Zuozhang; Xie, Lin; Han, Lei; Qu, Xin; Yang, Yihao; Zhang, Ya; He, Zewei; Wang, Yu; Li, Jing

2017-01-01

Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs featuring structural stability, high abundance, and tissue-specific expression. CircRNAs are prevalent and conserved in mammalian cells. They are involved in cellular processes and regulate gene expression at the transcriptional or post-transcriptional level by interacting with microRNAs (miRNAs) and other molecules. Recent studies have shown that circRNAs play an important role in the progression of various human diseases including atherosclerosis, nervous system disorders, diabetes, and cancer. In this review, we summarize the advances on endogenous circRNAs in eukaryotic cells and elucidate their diagnostic and prognostic significance in human cancers. Especially, we highlight the involvement of circRNAs in signal transduction pathways as well as their clinical potential to serve as biomarkers. PMID:28839467

Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization

PubMed Central

Cavaillé, Jérôme; Buiting, Karin; Kiefmann, Martin; Lalande, Marc; Brannan, Camilynn I.; Horsthemke, Bernhard; Bachellerie, Jean-Pierre; Brosius, Jürgen; Hüttenhofer, Alexander

2000-01-01

We have identified three C/D-box small nucleolar RNAs (snoRNAs) and one H/ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded in the brain-specific serotonin 2C receptor gene. The three human C/D box snoRNAs map to chromosome 15q11–q13, within a region implicated in the Prader–Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2′-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA. PMID:11106375

Prediction of bacterial small RNAs in the RsmA (CsrA) and ToxT pathways: a machine learning approach.

PubMed

Fakhry, Carl Tony; Kulkarni, Prajna; Chen, Ping; Kulkarni, Rahul; Zarringhalam, Kourosh

2017-08-22

Small RNAs (sRNAs) constitute an important class of post-transcriptional regulators that control critical cellular processes in bacteria. Recent research using high-throughput transcriptomic approaches has led to a dramatic increase in the discovery of bacterial sRNAs. However, it is generally believed that the currently identified sRNAs constitute a limited subset of the bacterial sRNA repertoire. In several cases, sRNAs belonging to a specific class are already known and the challenge is to identify additional sRNAs belonging to the same class. In such cases, machine-learning approaches can be used to predict novel sRNAs in a given class. In this work, we develop novel bioinformatics approaches that integrate sequence and structure-based features to train machine-learning models for the discovery of bacterial sRNAs. We show that features derived from recurrent structural motifs in the ensemble of low energy secondary structures can distinguish the RNA classes with high accuracy. We apply this approach to predict new members in two broad classes of bacterial small RNAs: 1) sRNAs that bind to the RNA-binding protein RsmA/CsrA in diverse bacterial species and 2) sRNAs regulated by the master regulator of virulence, ToxT, in Vibrio cholerae. The involvement of sRNAs in bacterial adaptation to changing environments is an increasingly recurring theme in current research in microbiology. It is likely that future research, combining experimental and computational approaches, will discover many more examples of sRNAs as components of critical regulatory pathways in bacteria. We have developed a novel approach for prediction of small RNA regulators in important bacterial pathways. This approach can be applied to specific classes of sRNAs for which several members have been identified and the challenge is to identify additional sRNAs.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

PubMed Central

Sierant, Malgorzata; Leszczynska, Grazyna; Sadowska, Klaudia; Dziergowska, Agnieszka; Rozanski, Michal; Sochacka, Elzbieta; Nawrot, Barbara

2016-01-01

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies. PMID:27566149

PIWI Proteins and PIWI-Interacting RNA: Emerging Roles in Cancer.

PubMed

Han, Yi-Neng; Li, Yuan; Xia, Sheng-Qiang; Zhang, Yuan-Yuan; Zheng, Jun-Hua; Li, Wei

2017-01-01

P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNAs (ncRNAs) and interact with PIWI proteins. piRNAs were primarily described in the germline, but emerging evidence revealed that piRNAs are expressed in a tissue-specific manner among multiple human somatic tissue types as well and play important roles in transposon silencing, epigenetic regulation, gene and protein regulation, genome rearrangement, spermatogenesis and germ stem-cell maintenance. PIWI proteins were first discovered in Drosophila and they play roles in spermatogenesis, germline stem-cell maintenance, self-renewal, retrotransposons silencing and the male germline mobility control. A growing number of studies have demonstrated that several piRNA and PIWI proteins are aberrantly expressed in various kinds of cancers and may probably serve as a novel biomarker and therapeutic target for cancer treatment. Nevertheless, their specific mechanisms and functions need further investigation. In this review, we discuss about the biogenesis, functions and the emerging role of piRNAs and PIWI proteins in cancer, providing novel insights into the possible applications of piRNAs and PIWI proteins in cancer diagnosis and clinical treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

PubMed

Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

2005-06-01

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

PubMed

Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

2014-03-28

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The Generation of Insulin Producing Cells from Human Mesenchymal Stem Cells by MiR-375 and Anti-MiR-9.

PubMed

Jafarian, Arefeh; Taghikani, Mohammad; Abroun, Saeid; Allahverdi, Amir; Lamei, Maryam; Lakpour, Niknam; Soleimani, Masoud

2015-01-01

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose. It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists.

PubMed

Ambrose, Ashley R; Alsahli, Mohammed A; Kurmani, Sameer A; Goodall, Alison H

2018-07-01

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r 2  > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r 2  > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.

Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq.

PubMed

Xie, Lin; Liao, Yedan; Shen, Lida; Hu, Fengdi; Yu, Sunlin; Zhou, Yonghong; Zhang, Ya; Yang, Yihao; Li, Dongqi; Ren, Minyan; Yuan, Zhongqin; Yang, Zuozhang

2017-06-27

Small cell osteosarcoma (SCO) is a rare subtype of osteosarcoma characterized by highly aggressive progression and a poor prognosis. The miRNA and mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) were obtained in 3 patients with SCO and 10 healthy individuals using high-throughput RNA-sequencing. We identified 37 dysregulated miRNAs and 1636 dysregulated mRNAs in patients with SCO compared to the healthy controls. Specifically, the 37 dysregulated miRNAs consisted of 27 up-regulated miRNAs and 10 down-regulated miRNAs; the 1636 dysregulated mRNAs consisted of 555 up-regulated mRNAs and 1081 down-regulated mRNAs. The target-genes of miRNAs were predicted, and 1334 negative correlations between miRNAs and mRNAs were used to construct an miRNA-mRNA regulatory network. Dysregulated genes were significantly enriched in pathways related to cancer, mTOR signaling and cell cycle signaling. Specifically, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p were significantly dysregulated miRNAs and exhibited a high degree of connectivity with target genes. Overall, the expression of dysregulated genes in tumor tissues and peripheral blood samples of patients with SCO measured by quantitative real-time polymerase chain reaction corroborated with our bioinformatics analyses based on the expression profiles of PBMCs from patients with SCO. Thus, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p may be involved in SCO tumorigenesis.

miRNAtools: Advanced Training Using the miRNA Web of Knowledge.

PubMed

Stępień, Ewa Ł; Costa, Marina C; Enguita, Francisco J

2018-02-16

Micro-RNAs (miRNAs) are small non-coding RNAs that act as negative regulators of the genomic output. Their intrinsic importance within cell biology and human disease is well known. Their mechanism of action based on the base pairing binding to their cognate targets have helped the development not only of many computer applications for the prediction of miRNA target recognition but also of specific applications for functional assessment and analysis. Learning about miRNA function requires practical training in the use of specific computer and web-based applications that are complementary to wet-lab studies. In order to guide the learning process about miRNAs, we have created miRNAtools (http://mirnatools.eu), a web repository of miRNA tools and tutorials. This article compiles tools with which miRNAs and their regulatory action can be analyzed and that function to collect and organize information dispersed on the web. The miRNAtools website contains a collection of tutorials that can be used by students and tutors engaged in advanced training courses. The tutorials engage in analyses of the functions of selected miRNAs, starting with their nomenclature and genomic localization and finishing with their involvement in specific cellular functions.

Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

PubMed Central

Khraiwesh, Basel; Zhu, Jian-Kang; Zhu, Jianhua

2011-01-01

Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. PMID:21605713

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

PubMed Central

Kim, Sang Hyon; Spensley, Mark; Choi, Seung Kook; Calixto, Cristiane P. G.; Pendle, Ali F.; Koroleva, Olga; Shaw, Peter J.; Brown, John W. S.

2010-01-01

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants. PMID:20081206

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.

PubMed

Smalheiser, Neil R; Lugli, Giovanni; Thimmapuram, Jyothi; Cook, Edwin H; Larson, John

2011-01-01

We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

PubMed Central

2013-01-01

Background Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. Methods miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. Results The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. Conclusion This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector. PMID:23924583

High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers of Kidney Damage in Patients with Systemic Lupus Erythematosus.

PubMed

Navarro-Quiroz, Elkin; Pacheco-Lugo, Lisandro; Lorenzi, Hernan; Díaz-Olmos, Yirys; Almendrales, Lisneth; Rico, Edwin; Navarro, Roberto; España-Puccini, Pierine; Iglesias, Antonio; Egea, Eduardo; Aroca, Gustavo

2016-01-01

Renal involvement is one of the most severe manifestations of systemic lupus erythematosus (SLE). Renal biopsy is the gold standard when it comes to knowing whether a patient has lupus nephritis, and the degree of renal disease present. However, the biopsy has various complications, bleeding being the most common. Therefore, the development of alternative, non-invasive diagnostic tests for kidney disease in patients with SLE is a priority. Micro RNAs (miRNAs) are differentially expressed in various tissues, and changes in their expression have been associated with several pathological processes. The aim of this study was to identify changes in the abundance of miRNAs in plasma samples from patients with lupus nephritis that could potentially allow the diagnosis of renal damage in SLE patients. This is an observational case-control cross-sectional study, in which we characterized the differential abundance profiles of miRNAs among patients with different degrees of lupus compared with SLE patients without renal involvement and healthy control individuals. We found 89 miRNAs with changes in their abundance between lupus nephritis patients and healthy controls, and 17 miRNAs that showed significant variations between SLE patients with or without renal involvement. Validation for qPCR of a group of miRNAs on additional samples from lupus patients with or without nephritis, and from healthy individuals, showed that five miRNAs presented an average detection sensitivity of 97%, a specificity of 70.3%, a positive predictive value of 82.5%, a negative predictive value of 96% and a diagnosis efficiency of 87.9%. These results strongly suggest that miR-221-5p, miR-380-3p, miR-556-5p, miR-758-3p and miR-3074-3p are potential diagnostic biomarkers of lupus nephritis in patients with SLE. The observed differential pattern of miRNA abundance may have functional implications in the pathophysiology of SLE renal damage.

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls

PubMed Central

2013-01-01

Background Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases. PMID:23327631

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls.

PubMed

Steudemann, Carola; Bauersachs, Stefan; Weber, Karin; Wess, Gerhard

2013-01-17

Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n=4) or suffering from DCM (n=4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p<0.05 and fold change (FC)>1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p>0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p>0.05). Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

PubMed

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

2013-12-23

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

MicroRNAs in right ventricular remodelling.

PubMed

Batkai, Sandor; Bär, Christian; Thum, Thomas

2017-10-01

Right ventricular (RV) remodelling is a lesser understood process of the chronic, progressive transformation of the RV structure leading to reduced functional capacity and subsequent failure. Besides conditions concerning whole hearts, some pathology selectively affects the RV, leading to a distinct RV-specific clinical phenotype. MicroRNAs have been identified as key regulators of biological processes that drive the progression of chronic diseases. The role of microRNAs in diseases affecting the left ventricle has been studied for many years, however there is still limited information on microRNAs specific to diseases in the right ventricle. Here, we review recently described details on the expression, regulation, and function of microRNAs in the pathological remodelling of the right heart. Recently identified strategies using microRNAs as pharmacological targets or biomarkers will be highlighted. Increasing knowledge of pathogenic microRNAs will finally help improve our understanding of underlying distinct mechanisms and help utilize novel targets or biomarkers to develop treatments for patients suffering from right heart diseases. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Quantitative Differential Expression Analysis Reveals Mir-7 As Major Islet MicroRNA

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Molano, R. Damaris; Pileggi, Antonello; Ricordi, Camillo; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2008-01-01

MicroRNAs (miRNAs) are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are associated with the acquisition and maintenance of a given phenotype, such as endocrine pancreas (islets). We hypothesized that a subset of miRNAs could be differentially expressed in the islets. Using miRNA microarray technology and quantitative RT-PCR we identified a subset of miRNAs that are the most differentially expressed islet miRNAs (ratio islet/acinar >150-fold), mir-7 being the most abundant. A similarly high ratio for mir-7 was observed in human islets. The ratio islet/acinar for mir-375, a previously described islet miRNA, was <10, and is 2.5X more abundant in the islets than mir-7. Therefore, we conclude that mir-7 is the most abundant endocrine miRNA in islets while mir-375 is the most abundant intra-islet miRNA. Our results may offer new insights into regulatory pathways of islet gene expression. PMID:18086561

[MicroRNAs: circulating biomarkers in type 2 Diabetes Mellitus and physical exercise].

PubMed

Gómez-Banoy, Nicolás; Mockus, Ismena

2016-03-01

MicroRNAs are small, non-coding molecules with a crucial function in the cell´s biologic regulation. Circulating levels of miRNAs may be useful biomarkers in metabolic diseases such as type 2 Diabetes Mellitus (DM2), which alters the circulating concentrations of several types of miRNA. Specific serum profiles of these molecules have been identified in high-risk patients before the development of DM2 and its chronic complications. Most importantly, these profiles can be modified with physical exercise, which is crucial in the treatment of metabolic diseases. Acute physical activity alone can induce changes in tissue specific miRNAs, and responses are different in aerobic or non-aerobic training. Muscle and cardiovascular miRNAs, which may play an important role in the adap tation to exercise, are predominantly altered. Even further, there is a correlation between serum levels of miRNAs and fitness, suggesting a role for chronic exercise in their regulation. Thus, miRNAs are molecules of growing importance in exercise physiology, and may be involved in the mechanisms behind the beneficial effects of physical activity for patients with metabolic diseases.

In vitro quantification of specific microRNA using molecular beacons

PubMed Central

Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2012-01-01

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples. PMID:22110035

The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

PubMed Central

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C.

2014-01-01

SUMMARY Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self mRNAs. We refer to this mechanism, which can prevent or reverse RNAe as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a trans-generational CSR-1 memory that recognizes and protects self mRNAs, allowing piRNAs to recognize foreign sequences innately, without need for prior exposure. PMID:24360782

Decoding the ubiquitous role of microRNAs in neurogenesis.

PubMed

Nampoothiri, Sreekala S; Rajanikant, G K

2017-04-01

Neurogenesis generates fledgling neurons that mature to form an intricate neuronal circuitry. The delusion on adult neurogenesis was far resolved in the past decade and became one of the largely explored domains to identify multifaceted mechanisms bridging neurodevelopment and neuropathology. Neurogenesis encompasses multiple processes including neural stem cell proliferation, neuronal differentiation, and cell fate determination. Each neurogenic process is specifically governed by manifold signaling pathways, several growth factors, coding, and non-coding RNAs. A class of small non-coding RNAs, microRNAs (miRNAs), is ubiquitously expressed in the brain and has emerged to be potent regulators of neurogenesis. It functions by fine-tuning the expression of specific neurogenic gene targets at the post-transcriptional level and modulates the development of mature neurons from neural progenitor cells. Besides the commonly discussed intrinsic factors, the neuronal morphogenesis is also under the control of several extrinsic temporal cues, which in turn are regulated by miRNAs. This review enlightens on dicer controlled switch from neurogenesis to gliogenesis, miRNA regulation of neuronal maturation and the differential expression of miRNAs in response to various extrinsic cues affecting neurogenesis.

Direct detection of microRNAs using isothermal amplification and molecular beacon with excellent sensitivity and specificity

NASA Astrophysics Data System (ADS)

Zhang, Wancun; Zhang, Qi; Qian, Zhiyu; Gu, Yueqing

2017-02-01

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

Ectopic expression of miR-126*, an intronic product of the vascular endothelial EGF-like 7 gene, regulates prostein translation and invasiveness of prostate cancer LNCaP cells.

PubMed

Musiyenko, Alla; Bitko, Vira; Barik, Sailen

2008-03-01

MicroRNAs (miRNAs) are endogenous noncoding RNAs that down-regulate gene expression by promoting cleavage or translational arrest of target mRNAs. While most miRNAs are transcribed from their own dedicated genes, some map to introns of 'host' transcripts, the biological significance of which remains unknown. Here, we show that prostate cells are naturally devoid of EGF-like domain 7 (Egfl7) transcripts and hence also deficient in a miRNA, miR-126*, generated from splicing and processing of its ninth intron. Use of recombinant and synthetic miRNAs or a specific antagomir established a role of miR-126* in silencing prostein in non-endothelial cells. We mapped two miR-126*-binding sites in the 3'UTR of the prostein mRNA required for translational repression. Transfection of synthetic miR-126* into prostate cancer LNCaP cells strongly reduced the translation of prostein. Interestingly, loss of prostein correlated with reduction of LNCaP cell migration and invasion. Thus, the robust expression of prostein protein in the prostate cells results from a combination of transcriptional activation of the prostein gene and absence of intronic miRNA-126* due to the prostate-specific repression of the Egfl7 gene. We conclude that intronic miRNAs from tissue-specific transcripts, or their natural absence, make cardinal contributions to cellular gene expression and phenotype. These findings also open the door to tissue-specific miRNA therapy.

The emergence of noncoding RNAs as Heracles in autophagy.

PubMed

Zhang, Jian; Wang, Peiyuan; Wan, Lin; Xu, Shouping; Pang, Da

2017-06-03

Macroautophagy/autophagy is a catabolic process that is widely found in nature. Over the past few decades, mounting evidence has indicated that noncoding RNAs, ranging from small noncoding RNAs to long noncoding RNAs (lncRNAs) and even circular RNAs (circRNAs), mediate the transcriptional and post-transcriptional regulation of autophagy-related genes by participating in autophagy regulatory networks. The differential expression of noncoding RNAs affects autophagy levels at different physiological and pathological stages, including embryonic proliferation and differentiation, cellular senescence, and even diseases such as cancer. We summarize the current knowledge regarding noncoding RNA dysregulation in autophagy and investigate the molecular regulatory mechanisms underlying noncoding RNA involvement in autophagy regulatory networks. Then, we integrate public resources to predict autophagy-related noncoding RNAs across species and discuss strategies for and the challenges of identifying autophagy-related noncoding RNAs. This article will deepen our understanding of the relationship between noncoding RNAs and autophagy, and provide new insights to specifically target noncoding RNAs in autophagy-associated therapeutic strategies.

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

PubMed Central

Enuka, Yehoshua; Lauriola, Mattia; Feldman, Morris E.; Sas-Chen, Aldema; Ulitsky, Igor; Yarden, Yosef

2016-01-01

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. PMID:26657629

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

PubMed Central

Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.

2010-01-01

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains supplementary material, which is available to authorized users. PMID:20145944

The specificity of long noncoding RNA expression.

PubMed

Gloss, Brian S; Dinger, Marcel E

2016-01-01

Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a fundamental molecular class whose members play pivotal roles in the regulation of the genome. The observation of pervasive transcription of mammalian genomes in the early 2000s sparked a revolution in the understanding of information flow in eukaryotic cells and the incredible flexibility and dynamic nature of the transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to outnumber those yielding mRNAs. However, like many important discoveries, the road leading to uncovering this diverse class of molecules that act through a remarkable repertoire of mechanisms, was not a straight one. The same characteristic that most distinguishes lncRNAs from mRNAs, i.e. their developmental-stage, tissue-, and cell-specific expression, was one of the major impediments to their discovery and recognition as potentially functional regulatory molecules. With growing numbers of lncRNAs being assigned to biological functions, the specificity of lncRNA expression is now increasingly recognized as a characteristic that imbues lncRNAs with great potential as biomarkers and for the development of highly targeted therapeutics. Here we review the history of lncRNA research and how technological advances and insight into biological complexity have gone hand-in-hand in shaping this revolution. We anticipate that as increasing numbers of these molecules, often described as the dark matter of the genome, are characterized and the structure-function relationship of lncRNAs becomes better understood, it may ultimately be feasible to decipher what these non-(protein)-coding genes encode. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

miRNA expression in control and FSHD fetal human muscle biopsies.

PubMed

Portilho, Débora Morueco; Alves, Marcelo Ribeiro; Kratassiouk, Gueorgui; Roche, Stéphane; Magdinier, Frédérique; de Santana, Eliane Corrêa; Polesskaya, Anna; Harel-Bellan, Annick; Mouly, Vincent; Savino, Wilson; Butler-Browne, Gillian; Dumonceaux, Julie

2015-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development. Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays. During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD. This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.

Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma

PubMed Central

Turchinovich, Andrey; Burwinkel, Barbara

2012-01-01

Studies of miRNA association with Argonaute (AGO) proteins in mammalian cells have indicated lack of bias toward particular AGO. However, to our knowledge, the use of quantitative methods for studying miRNA association with different AGOs has not been reported so far. In this work we compared the total miRNA content in AGO1 and AGO2 immunoprecipitates obtained from MCF7 adenocarcinoma cells using TaqMan Low Density miRNA Arrays and successfully verified selected miRNAs with qPCR. For most of the miRNA species AGO1 and AGO2 profiles were well correlated, however, some miRNAs demonstrated consistent biases toward one of the Argonautes. Furthermore, miRNAs which were predominantly AGO2-associated derived mostly from sense strands of the corresponding pre-miRNAs while the majority of AGO1 biased miRNAs originated from antisense strands of the pre-miRNAs. Additionally, we show that circulating miRNA in human blood plasma can be immunoprecipitated with both AGO1 and AGO2 antibody. However, unlike in cell lysates, AGO1 and AGO2 associated miRNA profiles in plasma did not correlate, indicating that many cell types contribute to circulating miRNA (given that expression of AGO proteins is tissue specific). Furthermore, AGO-specific miRNA profiles in blood cells differed significantly from miRNAs profiles in plasma indicating that most circulating miRNAs are likely to derive from non-blood cells. Since circulating miRNAs hold great promise as biomarkers for numerous cancers and other diseases, we hypothesize that AGO-specific miRNA profiles might add an additional dimension to circulating miRNA-based diagnostics. PMID:22858679

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Blood miRNAs as sensitive and specific biological indicators of environmental and occupational exposure to volatile organic compound (VOC).

PubMed

Song, Mi-Kyung; Ryu, Jae-Chun

2015-10-01

To date, there is still shortage of highly sensitive and specific minimally invasive biomarkers for assessment of environmental toxicants exposure. Because of the significance of microRNA (miRNA) in various diseases, circulating miRNAs in blood may be unique biomarkers for minimally invasive prediction of toxicants exposure. We identified and validated characteristic miRNA expression profiles of human whole blood in workers exposed to volatile organic compounds (VOCs) and compared the usefulness of miRNA indicator of VOCs with the effectiveness of the already used urinary biomarkers of occupational exposure. Using a microarray based approach we screened and detected deregulated miRNAs in their expression in workers exposed to VOCs (toluene [TOL], xylene [XYL] and ethylbenzene [EBZ]). Total 169 workers from four dockyards were enrolled in current study, and 50 subjects of them were used for miRNA microarray analysis. We identified 467 miRNAs for TOL, 211 miRNAs for XYL, and 695 miRNAs for XYL as characteristic discernible exposure indicator, which could discerned each VOC from the control group with higher accuracy, sensitivity, and specificity than urinary biomarkers. Current observations from this study point out that the altered levels of circulating miRNAs can be a reliable novel, minimally invasive biological indicator of occupational exposure to VOCs. Copyright © 2015 Elsevier GmbH. All rights reserved.

Human embryonic stem cells express a unique set of microRNAs.

PubMed

Suh, Mi-Ra; Lee, Yoontae; Kim, Jung Yeon; Kim, Soo-Kyoung; Moon, Sung-Hwan; Lee, Ji Yeon; Cha, Kwang-Yul; Chung, Hyung Min; Yoon, Hyun Soo; Moon, Shin Yong; Kim, V Narry; Kim, Kye-Seong

2004-06-15

Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.

Dicer-Like Proteins Regulate Sexual Development via the Biogenesis of Perithecium-Specific MicroRNAs in a Plant Pathogenic Fungus Fusarium graminearum

PubMed Central

Zeng, Wenping; Wang, Jie; Wang, Ying; Lin, Jing; Fu, Yanping; Xie, Jiatao; Jiang, Daohong; Chen, Tao; Liu, Huiquan; Cheng, Jiasen

2018-01-01

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development. PMID:29755439

RISC assembly: Coordination between small RNAs and Argonaute proteins.

PubMed

Kobayashi, Hotaka; Tomari, Yukihide

2016-01-01

Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors. Some steps of RISC assembly and RISC-mediated gene silencing are dependent on or facilitated by particular intracellular platforms, suggesting their spatial regulation. In this review, we summarize the currently known mechanisms for RISC assembly of each small RNA class and propose a revised model for the role of the chaperone machinery in the duplex-initiated RISC assembly pathway. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

Complete cure of persistent virus infections by antiviral siRNAs.

PubMed

Saulnier, Aure; Pelletier, Isabelle; Labadie, Karine; Colbère-Garapin, Florence

2006-01-01

Small interfering RNAs (siRNAs) have been developed as antiviral agents for mammalian cells. The capacity of specific siRNAs to prevent virus infections has been demonstrated, and there is evidence that these new antiviral agents could have a partial therapeutic effect a few days after infection. We investigated the possibility of curing a persistent infection, several months after becoming established, using an in vitro model of persistent poliovirus (PV) infection in HEp-2 cells. Despite high virus titers and the presence of PV mutants, repeated treatment with a mixture of two siRNAs targeting both noncoding and coding regions, one of them in a highly conserved region, resulted in the complete cure of the majority of persistently infected cultures. No escape mutants emerged in treated cultures. The antiviral effect of specific siRNAs, consistent with a mechanism of RNA interference, correlated with a decrease in the amount of viral RNA, until its complete disappearance, resulting in cultures cured of virions and viral RNA.

Exploring miRNA based approaches in cancer diagnostics and therapeutics.

PubMed

Mishra, Shivangi; Yadav, Tanuja; Rani, Vibha

2016-02-01

MicroRNAs (miRNAs), a highly conserved class of tissue specific, small non-protein coding RNAs maintain cell homeostasis by negative gene regulation. Proper controlling of miRNA expression is required for a balanced physiological environment, as these small molecules influence almost every genetic pathway from cell cycle checkpoint, cell proliferation to apoptosis, with a wide range of target genes. Deregulation in miRNAs expression correlates with various cancers by acting as tumor suppressors and oncogenes. Although promising therapies exist to control tumor development and progression, there is a lack of efficient diagnostic and therapeutic approaches for delineating various types of cancer. The molecularly different tumors can be differentiated by specific miRNA profiling as their phenotypic signatures, which can hence be exploited to surmount the diagnostic and therapeutic challenges. Present review discusses the involvement of miRNAs in oncogenesis with the analysis of patented research available on miRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization

PubMed Central

García-López, Jesús; Alonso, Lola; Cárdenas, David B.; Artaza-Alvarez, Haydeé; Hourcade, Juan de Dios; Martínez, Sergio; Brieño-Enríquez, Miguel A.; del Mazo, Jesús

2015-01-01

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes. PMID:25805854

Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes.

PubMed

Long, Tianyun; Lu, Rui

2017-01-01

Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

Zygotic amplification of secondary piRNAs during silkworm embryogenesis

PubMed Central

Kawaoka, Shinpei; Arai, Yuji; Kadota, Koji; Suzuki, Yutaka; Hara, Kahori; Sugano, Sumio; Shimizu, Kentaro; Tomari, Yukihide; Shimada, Toru; Katsuma, Susumu

2011-01-01

PIWI-interacting RNAs (piRNAs) are 23–30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle. PMID:21628432

RBM20 Regulates Circular RNA Production From the Titin Gene.

PubMed

Khan, Mohsin A F; Reckman, Yolan J; Aufiero, Simona; van den Hoogenhof, Maarten M G; van der Made, Ingeborg; Beqqali, Abdelaziz; Koolbergen, Dave R; Rasmussen, Torsten B; van der Velden, Jolanda; Creemers, Esther E; Pinto, Yigal M

2016-10-14

RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules. To establish the role of RBM20 in the formation of circRNAs in the heart. Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs. © 2016 American Heart Association, Inc.

Divergently expressed gene identification and interaction prediction of long noncoding RNA and mRNA involved in duck reproduction.

PubMed

Ren, Jindong; Du, Xue; Zeng, Tao; Chen, Li; Shen, Junda; Lu, Lizhi; Hu, Jianhong

2017-10-01

Long noncoding RNAs (lncRNAs) and divergently expressed genes exist widely in different tissues of mammals and birds, in which they are involved in various biological processes. However, there is limited information on their role in the regulation of normal biological processes during differentiation, development, and reproduction in birds. In this study, whole transcriptome strand-specific RNA sequencing of the ovary from young ducks (60days), first-laying ducks (160days), and old ducks, i.e., ducks that stopped laying eggs (490days) was performed. The lncRNAs and mRNAs from these ducks were systematically analyzed and identified by duck genome sequencing in the three study groups. The transcriptome from the duck ovary comprised 15,011 protein-coding genes and 2905 lncRNAs; all the lncRNAs were identified as novel long noncoding transcripts. The comparison of transcriptome data from different study groups identified 2240 divergent transcription genes and 135 divergently expressed lncRNAs, which differed among the groups; most of them were significantly downregulated with age. Among the divergent genes, 38 genes were related to the reproductive process and 6 genes were upregulated. Further prediction analysis revealed that 52 lncRNAs were closely correlated with divergent reproductive mRNAs. More importantly, 6 remarkable lncRNAs were correlated significantly with the conversion of the ovary in different phases. Our results aid in the understanding of the divergent transcriptome of duck ovary in different phases and the underlying mechanisms that drive the specificity of protein-coding genes and lncRNAs in duck ovary. Copyright © 2017. Published by Elsevier B.V.

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

PubMed Central

2011-01-01

Background Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. Results We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. Conclusion We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions. PMID:21812964

MicroRNAs Influence Reproductive Responses by Females to Male Sex Peptide in Drosophila melanogaster

PubMed Central

Fricke, Claudia; Green, Darrell; Smith, Damian; Dalmay, Tamas; Chapman, Tracey

2014-01-01

Across taxa, female behavior and physiology change significantly following the receipt of ejaculate molecules during mating. For example, receipt of sex peptide (SP) in female Drosophila melanogaster significantly alters female receptivity, egg production, lifespan, hormone levels, immunity, sleep, and feeding patterns. These changes are underpinned by distinct tissue- and time-specific changes in diverse sets of mRNAs. However, little is yet known about the regulation of these gene expression changes, and hence the potential role of microRNAs (miRNAs), in female postmating responses. A preliminary screen of genomic responses in females to receipt of SP suggested that there were changes in the expression of several miRNAs. Here we tested directly whether females lacking four of the candidate miRNAs highlighted (miR-279, miR-317, miR-278, and miR-184) showed altered fecundity, receptivity, and lifespan responses to receipt of SP, when mated once or continually to SP null or control males. The results showed that miRNA-lacking females mated to SP null males exhibited altered receptivity, but not reproductive output, in comparison to controls. However, these effects interacted significantly with the genetic background of the miRNA-lacking females. No significant survival effects were observed in miRNA-lacking females housed continually with SP null or control males. However, continual exposure to control males that transferred SP resulted in significantly higher variation in miRNA-lacking female lifespan than did continual exposure to SP null males. The results provide the first insight into the effects and importance of miRNAs in regulating postmating responses in females. PMID:25245794

Maternal And Neonatal Plasma MicroRNA Biomarkers For Fetal Alcohol Exposure In An Ovine Model

PubMed Central

Balaraman, Sridevi; Lunde, E. Raine; Sawant, Onkar; Cudd, Timothy A.; Washburn, Shannon E.; Miranda, Rajesh C.

2014-01-01

Background Plasma or circulating miRNAs (cirmiRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. Methods Pregnant sheep were exposed to a binge model of ethanol consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a three-trimester equivalent period from gestational day (GD) 4 to GD 132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte and leukocytes obtained from non-pregnant ewes, and plasma from pregnant ewes 24 hours following the last binge ethanol episode, and from newborn lambs, at birth on ~GD 147. Results Pregnant ewe and newborn lamb cirmiRNA profiles were similar to each other and different from non-pregnant female plasma, erythrocyte or leukocyte miRNAs. Significant changes in cirmiRNA profiles were observed in the ethanol-exposed ewe, and at birth, in the in utero, ethanol-exposed lamb. CirmiRNAs including miR-9, -15b, -19b and -20a were sensitive and specific measures of ethanol exposure in both pregnant ewe and newborn lamb. Additionally, ethanol exposure altered guide to passenger strand cirmiRNA ratios in the pregnant ewe, but not in the lamb. Conclusion Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. CirmiRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment. PMID:24588274

siRNA Versus miRNA as Therapeutics for Gene Silencing

PubMed Central

Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

2015-01-01

Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed. PMID:26372022

Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous Cyanobacteria.

PubMed

Brenes-Álvarez, Manuel; Olmedo-Verd, Elvira; Vioque, Agustín; Muro-Pastor, Alicia M

2016-01-01

Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated.

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features.

PubMed

Di, Chao; Yuan, Jiapei; Wu, Yue; Li, Jingrui; Lin, Huixin; Hu, Long; Zhang, Ting; Qi, Yijun; Gerstein, Mark B; Guo, Yan; Lu, Zhi John

2014-12-01

Recently, in addition to poly(A)+ long non-coding RNAs (lncRNAs), many lncRNAs without poly(A) tails, have been characterized in mammals. However, the non-polyA lncRNAs and their conserved motifs, especially those associated with environmental stresses, have not been fully investigated in plant genomes. We performed poly(A)- RNA-seq for seedlings of Arabidopsis thaliana under four stress conditions, and predicted lncRNA transcripts. We classified the lncRNAs into three confidence levels according to their expression patterns, epigenetic signatures and RNA secondary structures. Then, we further classified the lncRNAs to poly(A)+ and poly(A)- transcripts. Compared with poly(A)+ lncRNAs and coding genes, we found that poly(A)- lncRNAs tend to have shorter transcripts and lower expression levels, and they show significant expression specificity in response to stresses. In addition, their differential expression is significantly enriched in drought condition and depleted in heat condition. Overall, we identified 245 poly(A)+ and 58 poly(A)- lncRNAs that are differentially expressed under various stress stimuli. The differential expression was validated by qRT-PCR, and the signaling pathways involved were supported by specific binding of transcription factors (TFs), phytochrome-interacting factor 4 (PIF4) and PIF5. Moreover, we found many conserved sequence and structural motifs of lncRNAs from different functional groups (e.g. a UUC motif responding to salt and a AU-rich stem-loop responding to cold), indicated that the conserved elements might be responsible for the stress-responsive functions of lncRNAs. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa

PubMed Central

Morin, Ryan D.; Aksay, Gozde; Dolgosheina, Elena; Ebhardt, H. Alexander; Magrini, Vincent; Mardis, Elaine R.; Sahinalp, S. Cenk; Unrau, Peter J.

2008-01-01

The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, ∼21- and ∼24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/. PMID:18323537

Decoding Crucial LncRNAs Implicated in Neurogenesis and Neurological Disorders.

PubMed

Ayana, R; Singh, Shailja; Pati, Soumya

2017-04-15

Unraveling transcriptional heterogeneity and the labyrinthine nature of neurodevelopment can probe insights into neuropsychiatric disorders. It is noteworthy that adult neurogenesis is restricted to the subventricular and subgranular zones of the brain. Recent studies suggest long non-coding RNAs (lncRNAs) as an avant-garde class of regulators implicated in neurodevelopment. But, paucity exists in the knowledge regarding lncRNAs in neurogenesis and their associations with neurodevelopmental defects. To address this, we extensively reviewed the existing literature databases as well as performed relevant in-silico analysis. We utilized Allen Brain Atlas (ABA) differential search module and generated a catalogue of ∼30,000 transcripts specific to the neurogenic zones, including coding and non-coding transcripts. To explore the existing lncRNAs reported in neurogenesis, we performed extensive literature mining and identified 392 lncRNAs. These degenerate lncRNAs were mapped onto the ABA transcript list leading to detection of 20 lncRNAs specific to neurogenic zones (Dentate gyrus/Lateral ventricle), among which 10 showed associations to several neurodevelopmental disorders following in-silico mapping onto brain disease databases like Simons Foundation Autism Research Initiative, AutDB, and lncRNADisease. Notably, using ABA correlation module, we could establish lncRNA-to-mRNA coexpression networks for the above 10 candidate lncRNAs. Finally, pathway prediction revealed physical, biochemical, or regulatory interactions for nine lncRNAs. In addition, ABA differential search also revealed 54 novel significant lncRNAs from the null set (∼30,000). Conclusively, this review represents an updated catalogue of lncRNAs in neurogenesis and neurological diseases, and overviews the field of OMICs-based data analysis for understanding lncRNome-based regulation in neurodevelopment.

The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms.

PubMed

Drager, B J; Harkey, M A; Iwata, M; Whiteley, A H

1989-05-01

Adult tissues of the sea urchin, Strongylocentrotus purpuratus, were analyzed for the products of a set of genes whose expression, in the embryo, is restricted to the skeletogenic primary mesenchyme (PM). Three embryonic PM-specific mRNAs were found to be abundant in adult skeletal tissues (test and lantern), but not in a variety of soft tissues. Homologous mRNAs were also found in skeletal tissues of the congeneric sea urchin, S. droebachiensis, as well as a more distantly related echinoid, Dendraster excentricus, and an asteroid, Evasterias troschellii. The distributions of two of these RNAs were analyzed in regenerating spines of adult S. purpuratus using in situ hybridization. These gene products were localized primarily in the calcoblasts that accumulated at the regeneration site. In nonregenerating spines SpLM 18 RNAs, the most abundant of these gene products, were localized in a small population of noncalcoblast cells scattered through the spine shaft, and were absent from calcoblasts. These observations suggest that a program of gene expression associated with the process of calcification is conserved both developmentally through the period of metamorphosis and evolutionarily among the echinoderms.

Induction of specific neuron types by overexpression of single transcription factors.

PubMed

Teratani-Ota, Yusuke; Yamamizu, Kohei; Piao, Yulan; Sharova, Lioudmila; Amano, Misa; Yu, Hong; Schlessinger, David; Ko, Minoru S H; Sharov, Alexei A

2016-10-01

Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.

Perspectives of Long Non-Coding RNAs in Cancer Diagnostics

PubMed Central

Reis, Eduardo M.; Verjovski-Almeida, Sergio

2012-01-01

Long non-coding RNAs (lncRNAs) transcribed from intergenic and intronic regions of the human genome constitute a broad class of cellular transcripts that are under intensive investigation. While only a handful of lncRNAs have been characterized, their involvement in fundamental cellular processes that control gene expression highlights a central role in cell homeostasis. Not surprisingly, aberrant expression of regulatory lncRNAs has been increasingly documented in different types of cancer, where they can mediate both oncogenic or tumor suppressor effects. Interaction with chromatin remodeling complexes that promote silencing of specific genes or modulation of splicing factor proteins seem to be two general modes of lncRNA regulation, but it is conceivable that additional mechanisms of action are yet to be unveiled. LncRNAs show greater tissue specificity compared to protein-coding mRNAs making them attractive in the search of novel diagnostics/prognostics cancer biomarkers in body fluid samples. In fact, lncRNA prostate cancer antigen 3 can be detected in urine samples and has been shown to improve diagnosis of prostate cancer. We suggest that an unbiased screening of the presence of RNAs in easily accessible body fluids such as serum and urine might reveal novel circulating lncRNAs as potential biomarkers in many types of cancer. Annotation and functional characterization of the lncRNA complement of the cancer transcriptome will conceivably provide new venues for early diagnosis and treatment of the disease. PMID:22408643

The liverwort Pellia endiviifolia shares microtranscriptomic traits that are common to green algae and land plants

PubMed Central

Alaba, Sylwia; Piszczalka, Pawel; Pietrykowska, Halina; Pacak, Andrzej M; Sierocka, Izabela; Nuc, Przemyslaw W; Singh, Kashmir; Plewka, Patrycja; Sulkowska, Aleksandra; Jarmolowski, Artur; Karlowski, Wojciech M; Szweykowska-Kulinska, Zofia

2015-01-01

Liverworts are the most basal group of extant land plants. Nonetheless, the molecular biology of liverworts is poorly understood. Gene expression has been studied in only one species, Marchantia polymorpha. In particular, no microRNA (miRNA) sequences from liverworts have been reported. Here, Illumina-based next-generation sequencing was employed to identify small RNAs, and analyze the transcriptome and the degradome of Pellia endiviifolia. Three hundred and eleven conserved miRNA plant families were identified, and 42 new liverwort-specific miRNAs were discovered. The RNA degradome analysis revealed that target mRNAs of only three miRNAs (miR160, miR166, and miR408) have been conserved between liverworts and other land plants. New targets were identified for the remaining conserved miRNAs. Moreover, the analysis of the degradome permitted the identification of targets for 13 novel liverwort-specific miRNAs. Interestingly, three of the liverwort microRNAs show high similarity to previously reported miRNAs from Chlamydomonas reinhardtii. This is the first observation of miRNAs that exist both in a representative alga and in the liverwort P. endiviifolia but are not present in land plants. The results of the analysis of the P. endivifolia microtranscriptome support the conclusions of previous studies that placed liverworts at the root of the land plant evolutionary tree of life. PMID:25530158

MicroRNAs in Breastmilk and the Lactating Breast: Potential Immunoprotectors and Developmental Regulators for the Infant and the Mother

PubMed Central

Alsaweed, Mohammed; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

2015-01-01

Human milk (HM) is the optimal source of nutrition, protection and developmental programming for infants. It is species-specific and consists of various bioactive components, including microRNAs, small non-coding RNAs regulating gene expression at the post-transcriptional level. microRNAs are both intra- and extra-cellular and are present in body fluids of humans and animals. Of these body fluids, HM appears to be one of the richest sources of microRNA, which are highly conserved in its different fractions, with milk cells containing more microRNAs than milk lipids, followed by skim milk. Potential effects of exogenous food-derived microRNAs on gene expression have been demonstrated, together with the stability of milk-derived microRNAs in the gastrointestinal tract. Taken together, these strongly support the notion that milk microRNAs enter the systemic circulation of the HM fed infant and exert tissue-specific immunoprotective and developmental functions. This has initiated intensive research on the origin, fate and functional significance of milk microRNAs. Importantly, recent studies have provided evidence of endogenous synthesis of HM microRNA within the human lactating mammary epithelium. These findings will now form the basis for investigations of the role of microRNA in the epigenetic control of normal and aberrant mammary development, and particularly lactation performance. PMID:26529003

Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing

PubMed Central

Gao, Shuangcheng; Zhao, Wei; Li, Xiang; You, Qingbo; Shen, Xinjie; Guo, Wei; Wang, Shihua; Shi, Guoan; Liu, Zheng; Jiao, Yongqing

2017-01-01

Cleome gynandra and Cleome hassleriana, which are C4 and C3 plants, respectively, are two species of Cleome. The close genetic relationship between C. gynandra and C. hassleriana provides advantages for discovering the differences in leaf development and physiological processes between C3 and C4 plants. MicroRNAs (miRNAs) are a class of important regulators of various biological processes. In this study, we investigate the differences in the characteristics of miRNAs between C. gynandra and C. hassleriana using high-throughput sequencing technology. In total, 94 and 102 known miRNAs were identified in C. gynandra and C. hassleriana, respectively, of which 3 were specific for C. gynandra and 10 were specific for C. hassleriana. Ninety-one common miRNAs were identified in both species. In addition, 4 novel miRNAs were detected, including three in C. gynandra and three in C. hassleriana. Of these miRNAs, 67 were significantly differentially expressed between these two species and were involved in extensive biological processes, such as glycol-metabolism and photosynthesis. Our study not only provided resources for C. gynandra and C. hassleriana research but also provided useful clues for the understanding of the roles of miRNAs in the alterations of biological processes in leaf tissues during the evolution of the C4 pathway. PMID:28422166

Boron Stress Responsive MicroRNAs and Their Targets in Barley

PubMed Central

Ozhuner, Esma; Eldem, Vahap; Ipek, Arif; Okay, Sezer; Sakcali, Serdal; Zhang, Baohong; Boke, Hatice; Unver, Turgay

2013-01-01

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress. PMID:23555702

MicroRNA networks in mouse lung organogenesis.

PubMed

Dong, Jie; Jiang, Guoqian; Asmann, Yan W; Tomaszek, Sandra; Jen, Jin; Kislinger, Thomas; Wigle, Dennis A

2010-05-26

MicroRNAs (miRNAs) are known to be important regulators of both organ development and tumorigenesis. MiRNA networks and their regulation of messenger RNA (mRNA) translation and protein expression in specific biological processes are poorly understood. We explored the dynamic regulation of miRNAs in mouse lung organogenesis. Comprehensive miRNA and mRNA profiling was performed encompassing all recognized stages of lung development beginning at embryonic day 12 and continuing to adulthood. We analyzed the expression patterns of dynamically regulated miRNAs and mRNAs using a number of statistical and computational approaches, and in an integrated manner with protein levels from an existing mass-spectrometry derived protein database for lung development. In total, 117 statistically significant miRNAs were dynamically regulated during mouse lung organogenesis and clustered into distinct temporal expression patterns. 11,220 mRNA probes were also shown to be dynamically regulated and clustered into distinct temporal expression patterns, with 3 major patterns accounting for 75% of all probes. 3,067 direct miRNA-mRNA correlation pairs were identified involving 37 miRNAs. Two defined correlation patterns were observed upon integration with protein data: 1) increased levels of specific miRNAs directly correlating with downregulation of predicted mRNA targets; and 2) increased levels of specific miRNAs directly correlating with downregulation of translated target proteins without detectable changes in mRNA levels. Of 1345 proteins analyzed, 55% appeared to be regulated in this manner with a direct correlation between miRNA and protein level, but without detectable change in mRNA levels. Systematic analysis of microRNA, mRNA, and protein levels over the time course of lung organogenesis demonstrates dynamic regulation and reveals 2 distinct patterns of miRNA-mRNA interaction. The translation of target proteins affected by miRNAs independent of changes in mRNA level appears to be a prominent mechanism of developmental regulation in lung organogenesis.

Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

PubMed Central

Dalmay, Tamas

2018-01-01

RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues. PMID:29474379

Regulation of mammalian cell differentiation by long non-coding RNAs

PubMed Central

Hu, Wenqian; Alvarez-Dominguez, Juan R; Lodish, Harvey F

2012-01-01

Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non-coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non-coding species can modulate gene-expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non-coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non-coding RNAs contribute to the regulation of mammalian development. PMID:23070366

Human salivary microRNAs in Cancer

PubMed Central

Rapado-González, Óscar; Majem, Blanca; Muinelo-Romay, Laura; Álvarez-Castro, Ana; Santamaría, Anna; Gil-Moreno, Antonio; López-López, Rafael; Suárez-Cunqueiro, María Mercedes

2018-01-01

Circulating microRNAs (miRNAs) have emerged as excellent candidates for cancer biomarkers. Several recent studies have highlighted the potential use of saliva for the identification of miRNAs as novel biomarkers, which represents a great opportunity to improve diagnosis and monitor general health and disease. This review summarises the mechanisms of miRNAs deregulation in cancer, the value of targeting them with a therapeutic intention and the evidence of the potential clinical use of miRNAs expressed in saliva for the detection of different cancer types. We also provide a comprehensive review of the different methods for normalising the levels of specific miRNAs present in saliva, as this is a critical step in their analysis, and the challenge to validate salivary miRNAs as a reality to manage cancer patients. PMID:29556321

Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes

PubMed Central

Adir, Idan; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

2016-01-01

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal’s lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene’s promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs

PubMed Central

Ely, Abdullah; ul Islam, Rafique; Barichievy, Samantha; Bloom, Kristie; Weinberg, Marc S; van Otterlo, Willem AL; de Koning, Charles B; Salazar, Felix; Marion, Patricia; Roesch, Eric B; LeMaitre, Marc; Herdewijn, Piet

2010-01-01

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes. PMID:21687523

YamiPred: A Novel Evolutionary Method for Predicting Pre-miRNAs and Selecting Relevant Features.

PubMed

Kleftogiannis, Dimitrios; Theofilatos, Konstantinos; Likothanassis, Spiros; Mavroudi, Seferina

2015-01-01

MicroRNAs (miRNAs) are small non-coding RNAs, which play a significant role in gene regulation. Predicting miRNA genes is a challenging bioinformatics problem and existing experimental and computational methods fail to deal with it effectively. We developed YamiPred, an embedded classification method that combines the efficiency and robustness of support vector machines (SVM) with genetic algorithms (GA) for feature selection and parameters optimization. YamiPred was tested in a new and realistic human dataset and was compared with state-of-the-art computational intelligence approaches and the prevalent SVM-based tools for miRNA prediction. Experimental results indicate that YamiPred outperforms existing approaches in terms of accuracy and of geometric mean of sensitivity and specificity. The embedded feature selection component selects a compact feature subset that contributes to the performance optimization. Further experimentation with this minimal feature subset has achieved very high classification performance and revealed the minimum number of samples required for developing a robust predictor. YamiPred also confirmed the important role of commonly used features such as entropy and enthalpy, and uncovered the significance of newly introduced features, such as %A-U aggregate nucleotide frequency and positional entropy. The best model trained on human data has successfully predicted pre-miRNAs to other organisms including the category of viruses.

Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers.

PubMed

Cer, Regina Z; Herrera-Galeano, J Enrique; Frey, Kenneth G; Schully, Kevin L; Luu, Truong V; Pesce, John; Mokashi, Vishwesh P; Keane-Myers, Andrea M; Bishop-Lilly, Kimberly A

2017-01-01

Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5 α . RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis -exposed hPBMCs.

Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers

PubMed Central

Herrera-Galeano, J. Enrique; Frey, Kenneth G.; Schully, Kevin L.; Luu, Truong V.; Pesce, John; Mokashi, Vishwesh P.; Keane-Myers, Andrea M.; Bishop-Lilly, Kimberly A.

2017-01-01

Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs. PMID:28791299

The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets.

PubMed

Vakiloroayaei, Ana; Shah, Neha S; Oeffinger, Marlene; Bayfield, Mark A

2017-11-02

Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength.

PubMed

Manickam, Nandini; Joshi, Kartikeya; Bhatt, Monika J; Farabaugh, Philip J

2016-02-29

Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Understanding the Role of Non-Coding RNAs in Bladder Cancer: From Dark Matter to Valuable Therapeutic Targets

PubMed Central

Pop-Bica, Cecilia; Gulei, Diana; Cojocneanu-Petric, Roxana; Braicu, Cornelia; Petrut, Bogdan; Berindan-Neagoe, Ioana

2017-01-01

The mortality and morbidity that characterize bladder cancer compel this malignancy into the category of hot topics in terms of biomolecular research. Therefore, a better knowledge of the specific molecular mechanisms that underlie the development and progression of bladder cancer is demanded. Tumor heterogeneity among patients with similar diagnosis, as well as intratumor heterogeneity, generates difficulties in terms of targeted therapy. Furthermore, late diagnosis represents an ongoing issue, significantly reducing the response to therapy and, inevitably, the overall survival. The role of non-coding RNAs in bladder cancer emerged in the last decade, revealing that microRNAs (miRNAs) may act as tumor suppressor genes, respectively oncogenes, but also as biomarkers for early diagnosis. Regarding other types of non-coding RNAs, especially long non-coding RNAs (lncRNAs) which are extensively reviewed in this article, their exact roles in tumorigenesis are—for the time being—not as evident as in the case of miRNAs, but, still, clearly suggested. Therefore, this review covers the non-coding RNA expression profile of bladder cancer patients and their validated target genes in bladder cancer cell lines, with repercussions on processes such as proliferation, invasiveness, apoptosis, cell cycle arrest, and other molecular pathways which are specific for the malignant transformation of cells. PMID:28703782

Understanding the Role of Non-Coding RNAs in Bladder Cancer: From Dark Matter to Valuable Therapeutic Targets.

PubMed

Pop-Bica, Cecilia; Gulei, Diana; Cojocneanu-Petric, Roxana; Braicu, Cornelia; Petrut, Bogdan; Berindan-Neagoe, Ioana

2017-07-13

The mortality and morbidity that characterize bladder cancer compel this malignancy into the category of hot topics in terms of biomolecular research. Therefore, a better knowledge of the specific molecular mechanisms that underlie the development and progression of bladder cancer is demanded. Tumor heterogeneity among patients with similar diagnosis, as well as intratumor heterogeneity, generates difficulties in terms of targeted therapy. Furthermore, late diagnosis represents an ongoing issue, significantly reducing the response to therapy and, inevitably, the overall survival. The role of non-coding RNAs in bladder cancer emerged in the last decade, revealing that microRNAs (miRNAs) may act as tumor suppressor genes, respectively oncogenes, but also as biomarkers for early diagnosis. Regarding other types of non-coding RNAs, especially long non-coding RNAs (lncRNAs) which are extensively reviewed in this article, their exact roles in tumorigenesis are-for the time being-not as evident as in the case of miRNAs, but, still, clearly suggested. Therefore, this review covers the non-coding RNA expression profile of bladder cancer patients and their validated target genes in bladder cancer cell lines, with repercussions on processes such as proliferation, invasiveness, apoptosis, cell cycle arrest, and other molecular pathways which are specific for the malignant transformation of cells.

Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

PubMed Central

Ganapathi, Thumballi R.

2015-01-01

Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular. PMID:25962076

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE PAGES

Paugh, Steven W.; Coss, David R.; Bao, Ju; ...

2016-02-04

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paugh, Steven W.; Coss, David R.; Bao, Ju

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors. PMID:27171412

Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hudson, William H.; Pickard, Mark R.; de Vera, Ian Mitchelle S.

2014-12-23

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5more » lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.« less

Identification of microRNAs from Amur grape (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics.

PubMed

Wang, Chen; Han, Jian; Liu, Chonghuai; Kibet, Korir Nicholas; Kayesh, Emrul; Shangguan, Lingfei; Li, Xiaoying; Fang, Jinggui

2012-03-29

MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

Separate analysis of human papillomavirus E6 and E7 messenger RNAs to predict cervical neoplasia progression

PubMed Central

Liu, Shuling; Lachkar, Bouchra; Zhang, Shuang; Xu, Chenyang; Tenjimbayashi, Yuri; Shikama, Ayumi; Tasaka, Nobutaka; Akiyama, Azusa; Sakurai, Manabu; Nakao, Sari; Ochi, Hiroyuki; Onuki, Mamiko; Matsumoto, Koji; Yoshikawa, Hiroyuki; Satoh, Toyomi

2018-01-01

A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA (p = 0.00011, 0.80 and 0.54). The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA (p = 0.034, 0.73, 0.53, and 0.72). Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings. PMID:29466435

MicroRNAs – Important Molecules in Lung Cancer Research

PubMed Central

Leidinger, Petra; Keller, Andreas; Meese, Eckart

2011-01-01

MicroRNAs (miRNA) are important regulators of gene expression. They are involved in many physiological processes ensuring the cellular homeostasis of human cells. Alterations of the miRNA expression have increasingly been associated with pathophysiologic changes of cancer cells making miRNAs currently to one of the most analyzed molecules in cancer research. Here, we provide an overview of miRNAs in lung cancer. Specifically, we address biological functions of miRNAs in lung cancer cells, miRNA signatures generated from tumor tissue and from patients’ body fluids, the potential of miRNAs as diagnostic and prognostic biomarker for lung cancer, and its role as therapeutic target. PMID:22303398

Involvement of MicroRNAs in Lung Cancer Biology and Therapy

PubMed Central

Liu, Xi; Sempere, Lorenzo F.; Guo, Yongli; Korc, Murray; Kauppinen, Sakari; Freemantle, Sarah J.; Dmitrovsky, Ethan

2011-01-01

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. Expression profiles of specific miRNAs have improved cancer diagnosis and classification and even provided prognostic information in many human cancers, including lung cancer. Tumor suppressive and oncogenic miRNAs were uncovered in lung carcinogenesis. The biological functions of these miRNAs in lung cancer were recently validated in well characterized cellular, murine transgenic as well as transplantable lung cancer models and in human paired normal-malignant lung tissue banks and tissue arrays. Tumor suppressive and oncogenic miRNAs that were identified in lung cancer will be reviewed here. Emphasis is placed on highlighting those functionally validated miRNAs that are not only biomarkers of lung carcinogenesis, but also candidate pharmacologic targets. How these miRNA findings advance an understanding of lung cancer biology and could improve lung cancer therapy are discussed in this article. PMID:21420030

MicroRNAs in Palatogenesis and Cleft Palate

PubMed Central

Schoen, Christian; Aschrafi, Armaz; Thonissen, Michelle; Poelmans, Geert; Von den Hoff, Johannes W.; Carels, Carine E. L.

2017-01-01

Palatogenesis requires a precise spatiotemporal regulation of gene expression, which is controlled by an intricate network of transcription factors and their corresponding DNA motifs. Even minor perturbations of this network may cause cleft palate, the most common congenital craniofacial defect in humans. MicroRNAs (miRNAs), a class of small regulatory non-coding RNAs, have elicited strong interest as key regulators of embryological development, and as etiological factors in disease. MiRNAs function as post-transcriptional repressors of gene expression and are therefore able to fine-tune gene regulatory networks. Several miRNAs are already identified to be involved in congenital diseases. Recent evidence from research in zebrafish and mice indicates that miRNAs are key factors in both normal palatogenesis and cleft palate formation. Here, we provide an overview of recently identified molecular mechanisms underlying palatogenesis involving specific miRNAs, and discuss how dysregulation of these miRNAs may result in cleft palate. PMID:28420997

Long noncoding RNAs as enhancers of gene expression.

PubMed

Ørom, U A; Derrien, T; Guigo, R; Shiekhattar, R

2010-01-01

The human genome contains thousands of long noncoding RNAs (ncRNAs) transcribed from diverse genomic locations. A large set of long ncRNAs is transcribed independent of protein-coding genes. We have used the GENCODE annotation of the human genome to identify 3019 long ncRNAs expressed in various human cell lines and tissue. This set of long ncRNAs responds to differentiation signals in primary human keratinocytes and is coexpressed with important regulators of keratinocyte development. Depletion of a number of these long ncRNAs leads to the repression of specific genes in their surrounding locus, supportive of an activating function for ncRNAs. Using reporter assays, we confirmed such activating function and show that such transcriptional enhancement is mediated through the long ncRNA transcripts. Our studies show that long ncRNAs exhibit functions similar to classically defined enhancers, through an RNA-dependent mechanism.

Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

PubMed

Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

2016-09-07

Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of the Influence of microRNAs in Lithium Response in Bipolar Disorder.

PubMed

Reinbold, Céline S; Forstner, Andreas J; Hecker, Julian; Fullerton, Janice M; Hoffmann, Per; Hou, Liping; Heilbronner, Urs; Degenhardt, Franziska; Adli, Mazda; Akiyama, Kazufumi; Akula, Nirmala; Ardau, Raffaella; Arias, Bárbara; Backlund, Lena; Benabarre, Antonio; Bengesser, Susanne; Bhattacharjee, Abesh K; Biernacka, Joanna M; Birner, Armin; Marie-Claire, Cynthia; Cervantes, Pablo; Chen, Guo-Bo; Chen, Hsi-Chung; Chillotti, Caterina; Clark, Scott R; Colom, Francesc; Cousins, David A; Cruceanu, Cristiana; Czerski, Piotr M; Dayer, Alexandre; Étain, Bruno; Falkai, Peter; Frisén, Louise; Gard, Sébastien; Garnham, Julie S; Goes, Fernando S; Grof, Paul; Gruber, Oliver; Hashimoto, Ryota; Hauser, Joanna; Herms, Stefan; Jamain, Stéphane; Jiménez, Esther; Kahn, Jean-Pierre; Kassem, Layla; Kittel-Schneider, Sarah; Kliwicki, Sebastian; König, Barbara; Kusumi, Ichiro; Lackner, Nina; Laje, Gonzalo; Landén, Mikael; Lavebratt, Catharina; Leboyer, Marion; Leckband, Susan G; López Jaramillo, Carlos A; MacQueen, Glenda; Manchia, Mirko; Martinsson, Lina; Mattheisen, Manuel; McCarthy, Michael J; McElroy, Susan L; Mitjans, Marina; Mondimore, Francis M; Monteleone, Palmiero; Nievergelt, Caroline M; Ösby, Urban; Ozaki, Norio; Perlis, Roy H; Pfennig, Andrea; Reich-Erkelenz, Daniela; Rouleau, Guy A; Schofield, Peter R; Schubert, K Oliver; Schweizer, Barbara W; Seemüller, Florian; Severino, Giovanni; Shekhtman, Tatyana; Shilling, Paul D; Shimoda, Kazutaka; Simhandl, Christian; Slaney, Claire M; Smoller, Jordan W; Squassina, Alessio; Stamm, Thomas J; Stopkova, Pavla; Tighe, Sarah K; Tortorella, Alfonso; Turecki, Gustavo; Volkert, Julia; Witt, Stephanie H; Wright, Adam J; Young, L Trevor; Zandi, Peter P; Potash, James B; DePaulo, J Raymond; Bauer, Michael; Reininghaus, Eva; Novák, Tomáš; Aubry, Jean-Michel; Maj, Mario; Baune, Bernhard T; Mitchell, Philip B; Vieta, Eduard; Frye, Mark A; Rybakowski, Janusz K; Kuo, Po-Hsiu; Kato, Tadafumi; Grigoroiu-Serbanescu, Maria; Reif, Andreas; Del Zompo, Maria; Bellivier, Frank; Schalling, Martin; Wray, Naomi R; Kelsoe, John R; Alda, Martin; McMahon, Francis J; Schulze, Thomas G; Rietschel, Marcella; Nöthen, Markus M; Cichon, Sven

2018-01-01

Bipolar disorder (BD) is a common, highly heritable neuropsychiatric disease characterized by recurrent episodes of mania and depression. Lithium is the best-established long-term treatment for BD, even though individual response is highly variable. Evidence suggests that some of this variability has a genetic basis. This is supported by the largest genome-wide association study (GWAS) of lithium response to date conducted by the International Consortium on Lithium Genetics (ConLiGen). Recently, we performed the first genome-wide analysis of the involvement of miRNAs in BD and identified nine BD-associated miRNAs. However, it is unknown whether these miRNAs are also associated with lithium response in BD. In the present study, we therefore tested whether common variants at these nine candidate miRNAs contribute to the variance in lithium response in BD. Furthermore, we systematically analyzed whether any other miRNA in the genome is implicated in the response to lithium. For this purpose, we performed gene-based tests for all known miRNA coding genes in the ConLiGen GWAS dataset ( n = 2,563 patients) using a set-based testing approach adapted from the versatile gene-based test for GWAS (VEGAS2). In the candidate approach, miR-499a showed a nominally significant association with lithium response, providing some evidence for involvement in both development and treatment of BD. In the genome-wide miRNA analysis, 71 miRNAs showed nominally significant associations with the dichotomous phenotype and 106 with the continuous trait for treatment response. A total of 15 miRNAs revealed nominal significance in both phenotypes with miR-633 showing the strongest association with the continuous trait ( p = 9.80E-04) and miR-607 with the dichotomous phenotype ( p = 5.79E-04). No association between miRNAs and treatment response to lithium in BD in either of the tested conditions withstood multiple testing correction. Given the limited power of our study, the investigation of miRNAs in larger GWAS samples of BD and lithium response is warranted.

siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

PubMed

Vickers, Timothy A; Crooke, Stanley T

2012-07-01

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

Evaluation and control of miRNA-like off-target repression for RNA interference.

PubMed

Seok, Heeyoung; Lee, Haejeong; Jang, Eun-Sook; Chi, Sung Wook

2018-03-01

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago-miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

In the shadow: The emerging role of long non-coding RNAs in the immune response of Atlantic salmon.

PubMed

Tarifeño-Saldivia, E; Valenzuela-Miranda, D; Gallardo-Escárate, C

2017-08-01

The genomic era has increased the research effort to uncover how the genome of an organism, and specifically the transcriptome, is modulated after interplaying with pathogenic microorganisms and ectoparasites. However, the ever-increasing accessibility of sequencing technology has also evidenced regulatory roles of long non-coding RNAs (lncRNAs) related to several biological processes including immune response. This study reports a high-confidence annotation and a comparative transcriptome analysis of lncRNAs from several tissues of Salmo salar infected with the most prevalent pathogens in the Chilean salmon aquaculture such as the infectious salmon anemia (ISA) virus, the intracellular bacterium Piscirickettsia salmonis and the ectoparasite copepod Caligus rogercresseyi. Our analyses showed that lncRNAs are widely modulated during infection. However, this modulation is pathogen-specific and highly correlated with immuno-related genes associated with innate immune response. These findings represent the first discovery for the widespread differential expression of lncRNAs in response to infections with different types of pathogens in Atlantic salmon, suggesting that lncRNAs are pivotal player during the fish immune response. Copyright © 2017 Elsevier Ltd. All rights reserved.

The small RNA profile in latex from Hevea brasiliensis trees is affected by tapping panel dryness.

PubMed

Gébelin, Virginie; Leclercq, Julie; Kuswanhadi; Argout, Xavier; Chaidamsari, Tetty; Hu, Songnian; Tang, Chaorong; Sarah, Gautier; Yang, Meng; Montoro, Pascal

2013-10-01

Natural rubber is harvested by tapping Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. Harvesting stress can lead to tapping panel dryness (TPD). MicroRNAs (miRNAs) are induced by abiotic stress and regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs. This study set out to sequence miRNAs expressed in latex cells and to identify TPD-related putative targets. Deep sequencing of small RNAs was carried out on latex from trees affected by TPD using Solexa technology. The most abundant small RNA class size was 21 nucleotides for TPD trees compared with 24 nucleotides in healthy trees. By combining the LeARN pipeline, data from the Plant MicroRNA database and Hevea EST sequences, we identified 19 additional conserved and four putative species-specific miRNA families not found in previous studies on rubber. The relative transcript abundance of the Hbpre-MIR159b gene increased with TPD. This study revealed a small RNA-specific signature of TPD-affected trees. Both RNA degradation and a shift in miRNA biogenesis are suggested to explain the general decline in small RNAs and, particularly, in miRNAs.

Computational approach for elucidating interactions of cross-species miRNAs and their targets in Flaviviruses.

PubMed

Shinde, Santosh P; Banerjee, Amit Kumar; Arora, Neelima; Murty, U S N; Sripathi, Venkateswara Rao; Pal-Bhadra, Manika; Bhadra, Utpal

2015-03-01

Combating viral diseases has been a challenging task since time immemorial. Available molecular approaches are limited and not much effective for this daunting task. MicroRNA based therapies have shown promise in recent times. MicroRNAs are tiny non-coding RNAs that regulate translational repression of target mRNA in highly specific manner. In this study, we have determined the target regions for human and viral microRNAs in the conserved genomic regions of selected viruses of Flaviviridae family using miRanda and performed a comparative target selectivity analysis among them. Specific target regions were determined and they were compared extensively among themselves by exploring their position to determine the vicinity. Based on the multiplicity and cooperativity analysis, interaction maps were developed manually to represent the interactions between top-ranking miRNAs and genomes of the viruses considered in this study. Self-organizing map (SOM) was used to cluster the best-ranked microRNAs based on the vital physicochemical properties. This study will provide deep insight into the interrelation of the viral and human microRNAs interactions with the selected Flaviviridae genomes and will help to identify cross-species microRNA targets on the viral genome.

Noncoding RNA:RNA Regulatory Networks in Cancer

PubMed Central

Chan, Jia Jia; Tay, Yvonne

2018-01-01

Noncoding RNAs (ncRNAs) constitute the majority of the human transcribed genome. This largest class of RNA transcripts plays diverse roles in a multitude of cellular processes, and has been implicated in many pathological conditions, especially cancer. The different subclasses of ncRNAs include microRNAs, a class of short ncRNAs; and a variety of long ncRNAs (lncRNAs), such as lincRNAs, antisense RNAs, pseudogenes, and circular RNAs. Many studies have demonstrated the involvement of these ncRNAs in competitive regulatory interactions, known as competing endogenous RNA (ceRNA) networks, whereby lncRNAs can act as microRNA decoys to modulate gene expression. These interactions are often interconnected, thus aberrant expression of any network component could derail the complex regulatory circuitry, culminating in cancer development and progression. Recent integrative analyses have provided evidence that new computational platforms and experimental approaches can be harnessed together to distinguish key ceRNA interactions in specific cancers, which could facilitate the identification of robust biomarkers and therapeutic targets, and hence, more effective cancer therapies and better patient outcome and survival. PMID:29702599

Circular RNAs: A novel type of biomarker and genetic tools in cancer

PubMed Central

Han, Yi-Neng; Xia, Sheng-Qiang; Zhang, Yuan-Yuan; Zheng, Jun-Hua; Li, Wei

2017-01-01

Circular RNAs (circRNAs) are a novel type of universal and diverse endogenous noncoding RNAs (ncRNAs) and they form a covalently closed continuous loop without 5′ or 3′ tails unlike linear RNAs. Most circRNAs are presented with characteristics of abundance, stability, conservatism, and often exhibiting tissue/developmental-stage-specific expression. CircRNAs are generated either from exons or introns by back splicing or lariat introns. CircRNAs play important roles as miRNA sponges, gene transcription and expression regulators, RNA-binding protein (RBP) sponges and protein/peptide translators. Emerging evidence revealed the function of circRNAs in cancer and may potentially serve as a required novel biomarker and therapeutic target for cancer treatment. In this review, we discuss about the origins, characteristics and functions of circRNA and how they work as miRNA sponges, gene transcription and expression regulators, RBP sponges in cancer as well as current research methods of circRNAs, providing evidence for the significance of circRNAs in cancer diagnosis and clinical treatment. PMID:28969093

Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.).

PubMed

Wang, Yue; Xu, Tingting; He, Weiyi; Shen, Xiujing; Zhao, Qian; Bai, Jianlin; You, Minsheng

2018-01-01

Long non-coding RNAs (lncRNAs) are of particular interest because of their contributions to many biological processes. Here, we present the genome-wide identification and characterization of putative lncRNAs in a global insect pest, Plutella xylostella. A total of 8096 lncRNAs were identified and classified into three groups. The average length of exons in lncRNAs was longer than that in coding genes and the GC content was lower than that in mRNAs. Most lncRNAs were flanked by canonical splice sites, similar to mRNAs. Expression profiling identified 114 differentially expressed lncRNAs during the DBM development and found that majority were temporally specific. While the biological functions of lncRNAs remain uncharacterized, many are microRNA precursors or competing endogenous RNAs involved in micro-RNA regulatory pathways. This work provides a valuable resource for further studies on molecular bases for development of DBM and lay the foundation for discovery of lncRNA functions in P. xylostella. Copyright © 2017 Elsevier Inc. All rights reserved.

[Circular RNA in human disease and their potential clinic significance].

PubMed

Chen, Yonghua; Li, Cheng; Tan, Chunlu; Mai, Gang; Liu, Xubao

2017-02-10

Circular RNAs (circ RNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. They share a stable structure, high expression and often exhibit tissue/developmental-stage-specific expression. Emerging evidence indicates that circRNAs might play important roles in human disease, such as cancer, neurological disorders and atherosclerotic vascular disease risk. The huge potentials of circRNAs are recently being discovered from the laboratory to the clinic. CircRNAs might be developed as a potential novel and stable biomarker and potential drugs used in disease diagnosis and treatment. Here, we review the current understanding of the roles of circRNAs in human disease and their potential clinic significance in disease.

"Salivary exRNA biomarkers to detect gingivitis and monitor disease regression".

PubMed

Kaczor-Urbanowicz, Karolina Elżbieta; Trivedi, Harsh M; Lima, Patricia O; Camargo, Paulo M; Giannobile, William V; Grogan, Tristan R; Gleber-Netto, Frederico O; Whiteman, Yair; Li, Feng; Lee, Hyo Jung; Dharia, Karan; Aro, Katri; Carerras-Presas, Carmen Martin; Amuthan, Saarah; Vartak, Manjiri; Akin, David; Al-Adbullah, Hiba; Bembey, Kanika; Klokkevold, Perry R; Elashoff, David; Barnes, Virginia Monsul; Richter, Rose; DeVizio, William; Masters, James G; Wong, David

2018-05-19

This study tests the hypothesis that salivary extracellular RNA (exRNA) biomarkers can be developed for gingivitis detection and monitoring disease regression. Salivary exRNA biomarker candidates were developed from a total of 100 gingivitis and non-gingivitis individuals using Affymetrix's expression microarrays. The top ten differentially expressed exRNAs were tested in a clinical cohort to determine if the discovered salivary exRNA markers for gingivitis were associated with clinical gingivitis and disease regression. For this purpose, unstimulated saliva was collected from 30 randomly selected gingivitis subjects, the gingival and plaque indexes scores were taken at baseline, 3 & 6 weeks and salivary exRNAs were assayed by means of reverse transcription quantitative polymerase chain reaction. Eight salivary exRNA biomarkers developed for gingivitis were statistically significantly changed over time, consistent with disease regression. A panel of four salivary exRNAs [SPRR1A, lnc-TET3-2:1, FAM25A, CRCT1] can detect gingivitis with a clinical performance of 0.91 area under the curve (AUC), with 71% sensitivity and 100% specificity. The clinical values of the developed salivary exRNA biomarkers are associated with gingivitis regression. They offer strong potential to be advanced for definitive validation and clinical laboratory development test (LDT). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Unique and conserved microRNAs in wheat chromosome 5D revealed by next-generation sequencing.

PubMed

Kurtoglu, Kuaybe Yucebilgili; Kantar, Melda; Lucas, Stuart J; Budak, Hikmet

2013-01-01

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow-sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat.

Deregulated Cardiac Specific MicroRNAs in Postnatal Heart Growth.

PubMed

Yu, Pujiao; Wang, Hongbao; Xie, Yuan; Zhou, Jinzhe; Yao, Jianhua; Che, Lin

2016-01-01

The heart is recognized as an organ that is terminally differentiated by adulthood. However, during the process of human development, the heart is the first organ with function in the embryo and grows rapidly during the postnatal period. MicroRNAs (miRNAs, miRs), as regulators of gene expression, play important roles during the development of multiple systems. However, the role of miRNAs in postnatal heart growth is still unclear. In this study, by using qRT-PCR, we compared the expression of seven cardiac- or muscle-specific miRNAs that may be related to heart development in heart tissue from mice at postnatal days 0, 3, 8, and 14. Four miRNAs-miR-1a-3p, miR-133b-3p, miR-208b-3p, and miR-206-3p-were significantly decreased while miR-208a-3p was upregulated during the postnatal heart growth period. Based on these results, GeneSpring GX was used to predict potential downstream targets by performing a 3-way comparison of predictions from the miRWalk, PITA, and microRNAorg databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to identify potential functional annotations and signaling pathways related to postnatal heart growth. This study describes expression changes of cardiac- and muscle-specific miRNAs during postnatal heart growth and may provide new therapeutic targets for cardiovascular diseases.

Circulating microRNAs in Pancreatic Juice as Candidate Biomarkers of Pancreatic Cancer

PubMed Central

Wang, Jin; Raimondo, Massimo; Guha, Sushovan; Chen, Jinyun; Diao, Lixia; Dong, Xiaoqun; Wallace, Michael B.; Killary, Ann M.; Frazier, Marsha L.; Woodward, Timothy A.; Wang, Jing; Sen, Subrata

2014-01-01

Development of sensitive and specific biomarkers, preferably those circulating in body fluids is critical for early diagnosis of cancer. This study performed profiling of microRNAs (miRNAs) in exocrine pancreatic secretions (pancreatic juice) by microarray analysis utilizing pancreatic juice from 6 pancreatic ductal adenocarcinoma (PDAC) patients and two pooled samples from 6 non-pancreatic, non-healthy (NPNH) as controls. Differentially circulating miRNAs were subsequently validated in 88 pancreatic juice samples from 50 PDAC, 19 chronic pancreatitis (CP) patients and 19 NPNH controls. A marked difference in the profiles of four circulating miRNAs (miR-205, miR-210, miR-492, and miR-1427) was observed in pancreatic juice collected from patients with PDAC and those without pancreatic disease. Elevated levels of the four miRNAs together predicted PDAC with a specificity of 88% and sensitivity of 87%. Inclusion of serum CA19-9 level increased the sensitivity to 91% and the specificity to 100%. Enrichment of the four miRNAs in pancreatic juice was associated with decreased OS, as was the combination of miR-205 and miR-210. Higher contents of miR-205 and miR-210 were also associated with lymph node metastasis. Elevated levels of circulating miR-205, miR-210, miR-492, and miR-1247 in pancreatic juice are, therefore, promising candidate biomarkers of disease and poor prognosis in patients with PDAC. PMID:25258651

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

Characterization and potential role of microRNA in the Chinese dominant malaria mosquito Anopheles sinensis (Diptera: Culicidae) throughout four different life stages.

PubMed

Feng, Xinyu; Wu, Jiatong; Zhou, Shuisen; Wang, Jingwen; Hu, Wei

2018-01-01

microRNAs (miRNAs) are one kind of small non-coding RNAs widely distributed in insects. Many studies have shown that miRNAs play critical roles in development, differentiation, apoptosis, and innate immunity. However, there are a few reports describing miRNAs in Anopheles sinensis , the most common, and one of the dominant malaria mosquito in China. Here, we investigated the global miRNA expression profile across four different developmental stages including embryo, larval, pupal, and adult stages using Illumina Hiseq 2500 sequencing. In total, 164 miRNAs were obtained out of 107.46 million raw sequencing reads. 99 of them identified as known miRNAs, and the remaining 65 miRNAs were considered as novel. By analyzing the read counts of miRNAs in all developmental stages, 95 miRNAs showed stage-specific expression (q < 0.01 and |log2 (fold change)| > 1) in consecutive stages, indicating that these miRNAs may be involved in critical physiological activity during development. Sixteen miRNAs were identified to be commonly dysregulated throughout four developmental stages. Many miRNAs showed stage-specific expression, such as asi-miR-2943 was exclusively expressed in the embryo stage, and asi-miR-1891 could not be detected in larval stage. The expression of six selected differentially expressed miRNAs identified by qRT-PCR were consistent with our sequencing results. Furthermore, 5296 and 1902 target genes were identified for the dysregulated 68 known and 27 novel miRNAs respectively by combining miRanda and RNAhybrid prediction. GO annotation and KEGG pathway analysis for the predicted genes of dysregulated miRNAs revealed that they might be involved in a broad range of biological processes related with the development, such as membrane, organic substance transport and several key pathways including protein processing in endoplasmic reticulum, propanoate metabolism and folate biosynthesis. Thirty-two key miRNAs were identified by microRNA-gene network analysis. The present study represents the first global characterization of An. sinensis miRNAs in its four developmental stages. The presence and differential expression of An. sinensis miRNAs imply that such miRNAs may play critical roles in An. sinensis life cycle. A better understanding of the functions of these miRNAs will have great implication for the effective control of vector population and therefore interrupting malaria transmission.

Prediction of plant pre-microRNAs and their microRNAs in genome-scale sequences using structure-sequence features and support vector machine.

PubMed

Meng, Jun; Liu, Dong; Sun, Chao; Luan, Yushi

2014-12-30

MicroRNAs (miRNAs) are a family of non-coding RNAs approximately 21 nucleotides in length that play pivotal roles at the post-transcriptional level in animals, plants and viruses. These molecules silence their target genes by degrading transcription or suppressing translation. Studies have shown that miRNAs are involved in biological responses to a variety of biotic and abiotic stresses. Identification of these molecules and their targets can aid the understanding of regulatory processes. Recently, prediction methods based on machine learning have been widely used for miRNA prediction. However, most of these methods were designed for mammalian miRNA prediction, and few are available for predicting miRNAs in the pre-miRNAs of specific plant species. Although the complete Solanum lycopersicum genome has been published, only 77 Solanum lycopersicum miRNAs have been identified, far less than the estimated number. Therefore, it is essential to develop a prediction method based on machine learning to identify new plant miRNAs. A novel classification model based on a support vector machine (SVM) was trained to identify real and pseudo plant pre-miRNAs together with their miRNAs. An initial set of 152 novel features related to sequential structures was used to train the model. By applying feature selection, we obtained the best subset of 47 features for use with the Back Support Vector Machine-Recursive Feature Elimination (B-SVM-RFE) method for the classification of plant pre-miRNAs. Using this method, 63 features were obtained for plant miRNA classification. We then developed an integrated classification model, miPlantPreMat, which comprises MiPlantPre and MiPlantMat, to identify plant pre-miRNAs and their miRNAs. This model achieved approximately 90% accuracy using plant datasets from nine plant species, including Arabidopsis thaliana, Glycine max, Oryza sativa, Physcomitrella patens, Medicago truncatula, Sorghum bicolor, Arabidopsis lyrata, Zea mays and Solanum lycopersicum. Using miPlantPreMat, 522 Solanum lycopersicum miRNAs were identified in the Solanum lycopersicum genome sequence. We developed an integrated classification model, miPlantPreMat, based on structure-sequence features and SVM. MiPlantPreMat was used to identify both plant pre-miRNAs and the corresponding mature miRNAs. An improved feature selection method was proposed, resulting in high classification accuracy, sensitivity and specificity.

ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy

PubMed Central

Monga, Isha; Qureshi, Abid; Thakur, Nishant; Gupta, Amit Kumar; Kumar, Manoj

2017-01-01

Allele-specific siRNAs (ASP-siRNAs) have emerged as promising therapeutic molecules owing to their selectivity to inhibit the mutant allele or associated single-nucleotide polymorphisms (SNPs) sparing the expression of the wild-type counterpart. Thus, a dedicated bioinformatics platform encompassing updated ASP-siRNAs and an algorithm for the prediction of their inhibitory efficacy will be helpful in tackling currently intractable genetic disorders. In the present study, we have developed the ASPsiRNA resource (http://crdd.osdd.net/servers/aspsirna/) covering three components viz (i) ASPsiDb, (ii) ASPsiPred, and (iii) analysis tools like ASP-siOffTar. ASPsiDb is a manually curated database harboring 4543 (including 422 chemically modified) ASP-siRNAs targeting 78 unique genes involved in 51 different diseases. It furnishes comprehensive information from experimental studies on ASP-siRNAs along with multidimensional genetic and clinical information for numerous mutations. ASPsiPred is a two-layered algorithm to predict efficacy of ASP-siRNAs for fully complementary mutant (Effmut) and wild-type allele (Effwild) with one mismatch by ASPsiPredSVM and ASPsiPredmatrix, respectively. In ASPsiPredSVM, 922 unique ASP-siRNAs with experimentally validated quantitative Effmut were used. During 10-fold cross-validation (10nCV) employing various sequence features on the training/testing dataset (T737), the best predictive model achieved a maximum Pearson’s correlation coefficient (PCC) of 0.71. Further, the accuracy of the classifier to predict Effmut against novel genes was assessed by leave one target out cross-validation approach (LOTOCV). ASPsiPredmatrix was constructed from rule-based studies describing the effect of single siRNA:mRNA mismatches on the efficacy at 19 different locations of siRNA. Thus, ASPsiRNA encompasses the first database, prediction algorithm, and off-target analysis tool that is expected to accelerate research in the field of RNAi-based therapeutics for human genetic diseases. PMID:28696921

Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP

PubMed Central

Meier, Jan; Hovestadt, Volker; Zapatka, Marc; Pscherer, Armin; Lichter, Peter; Seiffert, Martina

2013-01-01

MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity. PMID:23673373

Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy

NASA Astrophysics Data System (ADS)

Li, Yan; Dai, Yu; Zhang, Xiaojin; Chen, Jihua

2017-07-01

MicroRNAs (miRNAs), small non-coding RNAs, play an important role in modulating cell proliferation, migration, and differentiation. Since miRNAs can regulate multiple cancer-related genes simultaneously, regulating miRNAs could target a set of related oncogenic genes or pathways. Owing to their reduced immune response and low toxicity, miRNAs with small size and low molecular weight have become increasingly promising therapeutic drugs in cancer therapy. However, one of the major challenges of miRNAs-based cancer therapy is to achieve specific, effective, and safe delivery of therapeutic miRNAs into cancer cells. Here we provide a strategy using three-layered polyplex with folic acid as a targeting group to systemically deliver miR-210 into breast cancer cells, which results in breast cancer growth being inhibited.

Role of microRNAs in herpesvirus latency and persistence.

PubMed

Grey, Finn

2015-04-01

The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections. © 2015 The Author.

Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy.

PubMed

Li, Yan; Dai, Yu; Zhang, Xiaojin; Chen, Jihua

2017-07-14

MicroRNAs (miRNAs), small non-coding RNAs, play an important role in modulating cell proliferation, migration, and differentiation. Since miRNAs can regulate multiple cancer-related genes simultaneously, regulating miRNAs could target a set of related oncogenic genes or pathways. Owing to their reduced immune response and low toxicity, miRNAs with small size and low molecular weight have become increasingly promising therapeutic drugs in cancer therapy. However, one of the major challenges of miRNAs-based cancer therapy is to achieve specific, effective, and safe delivery of therapeutic miRNAs into cancer cells. Here we provide a strategy using three-layered polyplex with folic acid as a targeting group to systemically deliver miR-210 into breast cancer cells, which results in breast cancer growth being inhibited.

Highly Complementary Target RNAs Promote Release of Guide RNAs from Human Argonaute2

PubMed Central

De, Nabanita; Young, Lisa; Lau, Pick-Wei; Meisner, Nicole-Claudia; Morrissey, David V.; MacRae, Ian J.

2013-01-01

SUMMARY Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This “unloading” activity can be enhanced by mismatches between the target and the guide 5′ end and attenuated by mismatches to the guide 3′ end. The introduction of 3′ mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3′ ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells. PMID:23664376

MicroRNAs regulate osteogenesis and chondrogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Shiwu, E-mail: shiwudong@gmail.com; Yang, Bo; Guo, Hongfeng

Highlights: Black-Right-Pointing-Pointer To focus on the role of miRNAs in chondrogenesis and osteogenesis. Black-Right-Pointing-Pointer Involved in the regulation of miRNAs in osteoarthritis. Black-Right-Pointing-Pointer To speculate some therapeutic targets for bone diseases. -- Abstract: MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potentialmore » gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.« less

Cancer cachexia-induced muscle atrophy: evidence for alterations in microRNAs important for muscle size.

PubMed

Lee, David E; Brown, Jacob L; Rosa-Caldwell, Megan E; Blackwell, Thomas A; Perry, Richard A; Brown, Lemuel A; Khatri, Bhuwan; Seo, Dongwon; Bottje, Walter G; Washington, Tyrone A; Wiggs, Michael P; Kong, Byung-Whi; Greene, Nicholas P

2017-05-01

Muscle atrophy is a hallmark of cancer cachexia resulting in impaired function and quality of life and cachexia is the immediate cause of death for 20-40% of cancer patients. Multiple microRNAs (miRNAs) have been identified as being involved in muscle development and atrophy; however, less is known specifically on miRNAs in cancer cachexia. The purpose of this investigation was to examine the miRNA profile of skeletal muscle atrophy induced by cancer cachexia to uncover potential miRNAs involved with this catabolic condition. Phosphate-buffered saline (PBS) or Lewis lung carcinoma cells (LLC) were injected into C57BL/6J mice at 8 wk of age. LLC animals were allowed to develop tumors for 4 wk to induce cachexia. Tibialis anterior muscles were extracted and processed to isolate small RNAs, which were used for miRNA sequencing. Sequencing results were assembled with mature miRNAs, and functions of miRNAs were analyzed by Ingenuity Pathway Analysis. LLC animals developed tumors that contributed to significantly smaller tibialis anterior muscles (18.5%) and muscle cross-sectional area (40%) compared with PBS. We found 371 miRNAs to be present in the muscle above background levels. Of these, nine miRNAs were found to be differentially expressed. Significantly altered groups of miRNAs were categorized into primary functionalities including cancer, cell-to-cell signaling, and cellular development among others. Gene network analysis predicted specific alterations of factors contributing to muscle size including Akt, FOXO3, and others. These results create a foundation for future research into the sufficiency of targeting these genes to attenuate muscle loss in cancer cachexia. Copyright © 2017 the American Physiological Society.

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Apple miRNAs and tasiRNAs with novel regulatory networks

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. PMID:22704043

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

PubMed

Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

2018-01-01

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tryndyak, Volodymyr P.; Latendresse, John R.; Montgomery, Beverly

MicroRNAs (miRNAs) are a class of small, conserved, tissue-specific regulatory non-coding RNAs that modulate a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, including nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the present study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used as potential biomarkers formore » the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically diverse strains of inbred male mice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were fed a choline- and folate-deficient (CFD) diet for 12 weeks. This diet induced liver injury in all mouse strains; however, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specific, with A/J, C57BL/6J, and C3H/HeJ mice being the least sensitive and WSB/EiJ mice being the most sensitive. The morphological changes in the livers were accompanied by differences in the levels of hepatic and plasma miRNAs. The levels of circulating miR-34a, miR-122, miR-181a, miR-192, and miR-200b miRNAs were significantly correlated with a severity of NAFLD-specific liver pathomorphological features, with the strongest correlation occurring with miR-34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD. -- Highlights: ► Choline- and folate-deficiency induces a strain-specific fatty liver injury in mice. ► The extent of liver pathology was accompanied by the changes in microRNA expression. ► The levels of circulating microRNAs mirror the magnitude of fatty liver injury. ► Plasma microRNAs may be sensitive noninvasive indicators of the fatty liver injury.« less

Bioinformatic identification and expression analysis of banana microRNAs and their targets.

PubMed

Chai, Juan; Feng, Renjun; Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

Bioinformatic Identification and Expression Analysis of Banana MicroRNAs and Their Targets

PubMed Central

Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions. PMID:25856313

Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells

PubMed Central

Pehserl, Anna-Maria; Ress, Anna Lena; Stanzer, Stefanie; Resel, Margit; Karbiener, Michael; Stadelmeyer, Elke; Stiegelbauer, Verena; Gerger, Armin; Mayr, Christian; Scheideler, Marcel; Hutterer, Georg C.; Bauernhofer, Thomas; Kiesslich, Tobias; Pichler, Martin

2016-01-01

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (>1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle–arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies. PMID:27916938

In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

PubMed Central

Dragon, François; Pogačić, Vanda; Filipowicz, Witold

2000-01-01

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins. PMID:10757788

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

PubMed Central

Karicheva, Olga Z.; Kolesnikova, Olga A.; Schirtz, Tom; Vysokikh, Mikhail Y.; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A.; Martin, Robert P.; Entelis, Nina; Tarassov, Ivan

2011-01-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNALeu(UUR). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNALeu(UUR) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders. PMID:21724600

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

PubMed

Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

2011-10-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

Modulations of RNA sequences by cytokinin in pumpkin cotyledons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, C.; Ertl, J.; Chen, C.

1987-04-01

Polyadenylated mRNAs from excised pumpkin cotyledons treated with or without 10/sup -4/ M benzyladenine (BA) for various time periods in suspension culture were assayed by in vitro translation in the presence of (/sup 35/S) methionine. The radioactive polypeptides were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Specific sequences of mRNAs were enhanced, reduced, induced, or suppressed by the hormone within 60 min of the application of BA to the cotyledons. Four independent cDNA clones of cytokinin-modulated mRNAs have been selected and characterized. RNA blot hybridization using the four cDNA probes also indicates that the levels of specific mRNAs aremore » modulated upward or downward by the hormone.« less

Application of small RNA technology for improved control of parasitic helminths.

PubMed

Britton, Collette; Winter, Alan D; Marks, Neil D; Gu, Henry; McNeilly, Tom N; Gillan, Victoria; Devaney, Eileen

2015-08-15

Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of miRNAs and siRNAs in post-transcriptional gene regulation in veterinary parasitic helminths and the potential value of these in parasite diagnosis and control. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Serum-circulating miRNAs predict neuroblastoma progression in mouse model of high-risk metastatic disease.

PubMed

Ramraj, Satish Kumar; Aravindan, Sheeja; Somasundaram, Dinesh Babu; Herman, Terence S; Natarajan, Mohan; Aravindan, Natarajan

2016-04-05

Circulating miRNAs have momentous clinical relevance as prognostic biomarkers and in the progression of solid tumors. Recognizing novel candidates of neuroblastoma-specific circulating miRNAs would allow us to identify potential prognostic biomarkers that could predict the switch from favorable to high-risk metastatic neuroblastoma (HR-NB). Utilizing mouse models of favorable and HR-NB and whole miRnome profiling, we identified high serum levels of 34 and low levels of 46 miRNAs in animals with HR-NB. Preferential sequence homology exclusion of mouse miRNAs identified 25 (11 increased; 14 decreased) human-specific prognostic marker candidates, of which, 21 were unique to HR-NB. miRNA QPCR validated miRnome profile. Target analysis defined the candidate miRNAs' signal transduction flow-through and demonstrated their converged roles in tumor progression. miRNA silencing studies verified the function of select miRNAs on the translation of at least 14 target proteins. Expressions of critical targets that correlate tumor progression in tissue of multifarious organs identify the orchestration of HR-NB. Significant (>10 fold) increase in serum levels of miR-381, miR-548h, and miR-580 identify them as potential prognostic markers for neuroblastoma progression. For the first time, we identified serum-circulating miRNAs that predict the switch from favorable to HR-NB and, further imply that these miRNAs could play a functional role in tumor progression.

Plasma viral microRNA profiles reveal potential biomarkers for chronic active Epstein-Barr virus infection.

PubMed

Kawano, Yoshihiko; Iwata, Seiko; Kawada, Jun-ichi; Gotoh, Kensei; Suzuki, Michio; Torii, Yuka; Kojima, Seiji; Kimura, Hiroshi; Ito, Yoshinori

2013-09-01

Chronic active Epstein-Barr virus (CAEBV) infection has high mortality and morbidity, and biomarkers for disease severity and prognosis are required. MicroRNAs (miRNAs) are small noncoding RNAs, and EBV encodes multiple miRNAs. Because plasma contains sufficiently stable miRNAs, circulating EBV-associated miRNA profiles were investigated as novel biomarkers in CAEBV infection. Plasma miRNA expression was assessed for 12 miRNAs encoded within 2 EBV open reading frames (BART and BHRF). Expression levels were investigated in 19 patients with CAEBV infection, 14 patients with infectious mononucleosis, and 11 healthy controls. Relative expression levels of plasma miRNAs were determined by TaqMan probe-based quantitative assay. Plasma miR-BART1-5p, 2-5p, 5, and 22 levels in patients with CAEBV infection were significantly greater than those in patients with infectious mononucleosis and in controls. Plasma miR-BART2-5p, 4, 7, 13, 15, and 22 levels were significantly elevated in patients with CAEBV infection with systemic symptoms, compared with levels in patients with no systemic symptoms. The levels of miR-BART2-5p, 13, and 15 showed clinical cutoff values associated with specific clinical conditions, in contrast to plasma EBV loads. Levels of specific plasma EBV miRNAs were elevated differentially in patients with CAEBV infection. Several EBV miRNAs, particularly miR-BART2-5p, 13, and 15, are potentially biomarkers of disease severity or prognosis.

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy.

PubMed

Jorge, Karina T O S; Souza, Renan P; Assis, Marieta T A; Araújo, Marcelo G; Locati, Massimo; Jesus, Amélia M R; Dias Baptista, Ida M F; Lima, Cristiano X; Teixeira, Antônio L; Teixeira, Mauro M; Soriani, Frederico M

2017-05-01

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy. Copyright © 2017 American Society for Microbiology.

The hot pepper (Capsicum annuum) microRNA transcriptome reveals novel and conserved targets: a foundation for understanding MicroRNA functional roles in hot pepper.

PubMed

Hwang, Dong-Gyu; Park, June Hyun; Lim, Jae Yun; Kim, Donghyun; Choi, Yourim; Kim, Soyoung; Reeves, Gregory; Yeom, Seon-In; Lee, Jeong-Soo; Park, Minkyu; Kim, Seungill; Choi, Ik-Young; Choi, Doil; Shin, Chanseok

2013-01-01

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. Here, we employed high-throughput sequencing technology to identify miRNAs in pepper extensively from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on a bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Northern blot analysis was used to validate further the expression of representative miRNAs and to analyze their tissue-specific or developmental stage-specific expression patterns. Moreover, we computationally predicted miRNA targets, many of which were experimentally confirmed using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR-396 was a domain rearranged methyltransferase, the major de novo methylation enzyme, involved in RNA-directed DNA methylation in plants. This work provides the first reliable draft of the pepper miRNA transcriptome. It offers an expanded picture of pepper miRNAs in relation to other plants, providing a basis for understanding the functional roles of miRNAs in pepper.

Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts.

PubMed

Müller, Gerd A; Hansen, Uwe; Xu, Zhi; Griswold, Benjamin; Talan, Mark I; McDonnell, Nazli B; Briest, Wilfried

2012-02-01

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.

Comparative MicroRNA Expression Patterns in Fibroblasts after Low and High Doses of Low-LET Radiation Exposure

NASA Technical Reports Server (NTRS)

Maes, Olivier C.; Xu, Suying; Hada, Megumi; Wu, Honglu; Wang, Eugenia

2007-01-01

Exposure to ionizing radiation causes DNA damage to cells, and provokes a plethora of cellular responses controlled by unique gene-directed signaling pathways. MicroRNAs (miRNAs) are small (22-nucleotide), non-coding RNAs which functionally silence gene expression by either degrading the messages or inhibiting translation. Here we investigate radiation-dependent changes in these negative regulators by comparing the expression patterns of all 462 known human miRNAs in fibroblasts, after exposure to low (0.1 Gy) or high (2 Gy) doses of X-rays at 30 min, 2, 6 and 24 hrs post-treatment. The expression patterns of microRNAs after low and high doses of radiation show a similar qualitative down-regulation trend at early (0.5 hr) and late (24 hr) time points, with a quantitatively steeper slope following the 2 Gy exposures. Interestingly, an interruption of this downward trend is observed after the 2 Gy exposure, i.e. a significant up-regulation of microRNAs at 2 hrs, then reverting to the downward trend by 6 hrs; this interruption at the intermediate time point was not observed with the 0.1 Gy exposure. At the early time point (0.5 hr), candidate gene targets of selected down-regulated microRNAs, common to both 0.1 and 2 Gy exposures, were those functioning in chromatin remodeling. Candidate target genes of unique up-regulated microRNAs seen at a 2 hr intermediate time point, after the 2 Gy exposure only, are those involved in cell death signaling. Finally, putative target genes of down-regulated microRNAs seen at the late (24 hr) time point after either doses of radiation are those involved in the up-regulation of DNA repair, cell signaling and homeostasis. Thus we hypothesize that after radiation exposure, microRNAs acting as hub negative regulators for unique signaling pathways needed to be down-regulated so as to de-repress their target genes for the proper cellular responses, including DNA repair and cell maintenance. The unique microRNAs up-regulated at 2 hr after 2 Gy suggest the cellular response to functionally suppress the apoptotic death signaling reflex after exposure to high dose radiation. Further analyses with transcriptome and global proteomic profiling will validate the reciprocal expression of signature microRNAs selected in our radiation-exposed cells, and their candidate target gene families, and test our hypothesis that unique radiation-specific microRNAs are keys in governing signaling responses for damage control of this environmental hazard.

Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

Serum miRNAs miR-23a, 206, and 499 as Potential Biomarkers for Skeletal Muscle Atrophy

PubMed Central

Wang, Jing; He, Jian; Li, Wenjiong; Li, Jinglong; Chen, Shengju; Zhang, Peng; Liu, Hongju

2017-01-01

Muscle biopsy has long been expected to be replaced by noninvasive biomarkers with diagnostic value and prognostic applications for muscle atrophy. Growing evidence suggests that circulating microRNAs (miRNAs) could act as biomarkers for numerous pathophysiological statuses. In the present study, our results showed that the serum levels of six muscle-specific miRNAs (miR-1/23a/133/206/208b/499) were all elevated in unloading induced mice. The medium levels of these six muscle-specific miRNAs were all elevated in starvation induced atrophic C2C12 myotubes. Moreover, the serum levels of miR-23a/206/499 were induced in participants after 45 days of head-down bed rest (HDBR). The levels of miR-23a/206/499 were positively correlated with the ratio of soleus volume loss in HDBR participants, indicating that they might represent the process of muscle loss. In conclusion, our results demonstrated that circulating miRNAs could serve as useful biochemical and molecular indicators for muscle atrophy diagnosis and disease progression. PMID:29214178

miR-133 regulates Evi1 expression in AML cells as a potential therapeutic target.

PubMed

Yamamoto, Haruna; Lu, Jun; Oba, Shigeyoshi; Kawamata, Toyotaka; Yoshimi, Akihide; Kurosaki, Natsumi; Yokoyama, Kazuaki; Matsushita, Hiromichi; Kurokawa, Mineo; Tojo, Arinobu; Ando, Kiyoshi; Morishita, Kazuhiro; Katagiri, Koko; Kotani, Ai

2016-01-12

The Ecotropic viral integration site 1 (Evi1) is a zinc finger transcription factor, which is located on chromosome 3q26, over-expression in some acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Elevated Evi1 expression in AML is associated with unfavorable prognosis. Therefore, Evi1 is one of the strong candidate in molecular target therapy for the leukemia. MicroRNAs (miRNAs) are small non-coding RNAs, vital to many cell functions that negatively regulate gene expression by translation or inducing sequence-specific degradation of target mRNAs. As a novel biologics, miRNAs is a promising therapeutic target due to its low toxicity and low cost. We screened miRNAs which down-regulate Evi1. miR-133 was identified to directly bind to Evi1 to regulate it. miR-133 increases drug sensitivity specifically in Evi1 expressing leukemic cells, but not in Evi1-non-expressing cells The results suggest that miR-133 can be promising therapeutic target for the Evi1 dysregulated poor prognostic leukemia.

Harnessing RNA interference to develop neonatal therapies: from Nobel Prize winning discovery to proof of concept clinical trials.

PubMed

DeVincenzo, John P

2009-10-01

A revolution in the understanding of RNA biological processing and control is leading to revolutionary new concepts in human therapeutics. It has become increasingly clear that the so called "non-coding RNA" exerts specific and profound functional control on regulation of protein production and indeed controls the expression of all genes. Harnessing this naturally-occurring RNA-mediated regulation of protein production has immense human therapeutic potential. These processes are collectively known as RNA interference (RNAi). RNAi is a recently discovered, naturally-occurring intracellular process that regulates gene expression through the silencing of specific mRNAs. Methods of harnessing this natural pathway are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small inhibitory RNAs (siRNA). siRNAs are being chemically modified to acquire drug-like properties. Numerous recent high profile publications have provided proofs of concept that RNA interference may be useful therapeutically. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. This makes this approach very attractive for developing therapies targeting orphan diseases including neonatal diseases. Theoretically, any disease that can be ameliorated through knockdown of any endogenous or exogenous protein is a potential therapeutic target for RNAi-based therapeutics. Lung diseases are particularly attractive targets for RNAi therapeutics since the affected cells' location increases their accessibility to topical administration of siRNA, for example by aerosol. Respiratory viral infections and chronic lung disease are examples of such diseases. RNAi therapeutics have been shown to be active against RSV, parainfluenza and human metapneumoviruses in vitro and in vivo resulting in profound antiviral effects. The first proof of concept test of efficacy of an RNAi-based therapeutic in man has been initiated. A discussion of the science behind RNA interference is followed by a presentation of the potential practical issues in applying this technology to neonatal respiratory viral diseases. RNAi may offer new strategies for the treatment of a variety of orphan diseases including neonatal diseases, RSV infections, and other respiratory viruses.

Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. Results We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. Conclusions This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1]. PMID:23163954

MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.

PubMed

Baier, Scott R; Nguyen, Christopher; Xie, Fang; Wood, Jennifer R; Zempleni, Janos

2014-10-01

MicroRNAs (miRNAs) regulate genes in animals and plants and can be synthesized endogenously. In milk, miRNAs are encapsulated in exosomes, thereby conferring protection against degradation and facilitating uptake by endocytosis. The majority of bovine miRNAs have nucleotide sequences complementary to human gene transcripts, suggesting that miRNAs in milk might regulate human genes. We tested the hypotheses that humans absorb biologically meaningful amounts of miRNAs from nutritionally relevant doses of milk, milk-borne miRNAs regulate human gene expression, and mammals cannot compensate for dietary miRNA depletion by endogenous miRNA synthesis. Healthy adults (3 men, 2 women; aged 26-49 y) consumed 0.25, 0.5, and 1.0 L of milk in a randomized crossover design. Gene expression studies and milk miRNA depletion studies were conducted in human cell cultures and mice, respectively. For comparison, feeding studies with plant miRNAs from broccoli were conducted in humans. Postprandial concentration time curves suggest that meaningful amounts of miRNA (miR)-29b and miR-200c were absorbed; plasma concentrations of miR-1 did not change (negative control). The expression of runt-related transcription factor 2 (RUNX2), a known target of miR-29b, increased by 31% in blood mononuclear cells after milk consumption compared with baseline. When milk exosomes were added to cell culture media, mimicking postprandial concentrations of miR-29b and miR-200c, reporter gene activities significantly decreased by 44% and 17%, respectively, compared with vehicle controls in human embryonic kidney 293 cells. When C57BL/6J mice were fed a milk miRNA-depleted diet for 4 wk, plasma miR-29b concentrations were significantly decreased by 61% compared with miRNA-sufficient controls, i.e., endogenous synthesis did not compensate for dietary depletion. Broccoli sprout feeding studies were conducted as a control and elicited no detectable increase in Brassica-specific miRNAs. We conclude that miRNAs in milk are bioactive food compounds that regulate human genes. © 2014 American Society for Nutrition.

A tool for design of primers for microRNA-specific quantitative RT-qPCR.

PubMed

Busk, Peter K

2014-01-28

MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets. I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies. The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance

PubMed Central

Leidinger, Petra; Backes, Christina; Rheinheimer, Stefanie; Keller, Andreas; Meese, Eckart

2015-01-01

Background Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays. Results While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding. Conclusion The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions. PMID:26599228

In vivo delivery of miRNAs for cancer therapy: Challenges and strategies⋆

PubMed Central

Chen, Yunching; Gao, Dong-Yu; Huang, Leaf

2016-01-01

MicroRNAs (miRNAs), small non-coding RNAs, can regulate post-transcriptional gene expressions and silence a broad set of target genes. miRNAs, aberrantly expressed in cancer cells, play an important role in modulating gene expressions, thereby regulating downstream signaling pathways and affecting cancer formation and progression. Oncogenes or tumor suppressor genes regulated by miRNAs mediate cell cycle progression, metabolism, cell death, angiogenesis, metastasis and immunosuppression in cancer. Recently, miRNAs have emerged as therapeutic targets or tools and biomarkers for diagnosis and therapy monitoring in cancer. Since miRNAs can regulate multiple cancer-related genes simultaneously, using miRNAs as a therapeutic approach plays an important role in cancer therapy. However, one of the major challenges of miRNA-based cancer therapy is to achieve specific, efficient and safe systemic delivery of therapeutic miRNAs In vivo. This review discusses the key challenges to the development of the carriers for miRNA-based therapy and explores current strategies to systemically deliver miRNAs to cancer without induction of toxicity. PMID:24859533

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

PubMed Central

Motorin, Y; Grosjean, H

1999-01-01

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w. PMID:10445884

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

PubMed Central

Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

2016-01-01

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

Identification of human microRNA targets from isolated argonaute protein complexes.

PubMed

Beitzinger, Michaela; Peters, Lasse; Zhu, Jia Yun; Kremmer, Elisabeth; Meister, Gunter

2007-06-01

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ounzain, Samir; Pezzuto, Iole; Micheletti, Rudi

We report here that the key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Throughmore » a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.« less

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease

DOE PAGES

Ounzain, Samir; Pezzuto, Iole; Micheletti, Rudi; ...

2014-08-19

We report here that the key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Throughmore » a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.« less

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs

PubMed Central

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G.; Rigoutsos, Isidore

2017-01-01

Abstract Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. PMID:28108659

MicroRNA Biomarkers of Toxicity in Biological Matrices ...

EPA Pesticide Factsheets

Biomarker measurements that reliably correlate with tissue injury and can be measured from sampling accessible biofluids offer enormous benefits in terms of cost, time, and convenience when assessing environmental and drug-induced toxicity in model systems or human cohorts. MicroRNAs (miRNAs) have emerged in recent years as a promising new type of biomarker for monitoring toxicity. Recent enthusiasm for miRNA biomarker research has been fueled by discoveries that certain miRNA species are cell-type specific and released during injury, thus raising the possibility of using biofluid-based miRNAs as a “liquid biopsy” that may be obtained by sampling extracellular fluids. As biomarkers, miRNAs demonstrate improved stability as compared to many protein markers and sequences are largely conserved across species, simplifying analytical techniques. Recent efforts have sought to identify miRNAs that are released into accessible biofluids following xenobiotic exposure, using compounds that target specific organs. While still early in the discovery phase, miRNA biomarkers will have an increasingly important role in the assessment of adverse effects of both environmental chemicals and pharmaceutical drugs. Here, we review the current findings of biofluid-based miRNAs, as well as highlight technical challenges in assessing toxicologic pathology using these biomarkers. MicroRNAs (miRNAs) are small, non-coding RNA species that selectively bind mRNA molecules and alter thei

Influence of physical exercise on microRNAs in skeletal muscle regeneration, aging and diseases

PubMed Central

Ultimo, Simona; Zauli, Giorgio; Martelli, Alberto M.; Vitale, Marco; McCubrey, James A.; Capitani, Silvano; Neri, Luca M.

2018-01-01

Skeletal muscle is a dynamic tissue with remarkable plasticity and its growth and regeneration are highly organized, with the activation of specific transcription factors, proliferative pathways and cytokines. The decline of skeletal muscle tissue with age, is one of the most important causes of functional loss of independence in older adults. Maintaining skeletal muscle function throughout the lifespan is a prerequisite for good health and independent living. Physical activity represents one of the most effective preventive agents for muscle decay in aging. Several studies have underlined the importance of microRNAs (miRNAs) in the control of myogenesis and of skeletal muscle regeneration and function. In this review, we reported an overview and recent advances about the role of miRNAs expressed in the skeletal muscle, miRNAs regulation by exercise in skeletal muscle, the consequences of different physical exercise training modalities in the skeletal muscle miRNA profile, their regulation under pathological conditions and the role of miRNAs in age-related muscle wasting. Specific miRNAs appear to be involved in response to different types of exercise and therefore to play an important role in muscle fiber identity and myofiber gene expression in adults and elder population. Understanding the roles and regulation of skeletal muscle miRNAs during muscle regeneration may result in new therapeutic approaches in aging or diseases with impaired muscle function or re-growth. PMID:29682218

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Circulating microRNAs as novel biomarkers for bone diseases - Complex signatures for multifactorial diseases?

PubMed

Hackl, Matthias; Heilmeier, Ursula; Weilner, Sylvia; Grillari, Johannes

2016-09-05

Biomarkers are essential tools in clinical research and practice. Useful biomarkers must combine good measurability, validated association with biological processes or outcomes, and should support clinical decision making if used in clinical practice. Several types of validated biomarkers have been reported in the context of bone diseases. However, because these biomarkers face certain limitations there is an interest in the identification of novel biomarkers for bone diseases, specifically in those that are tightly linked to the disease pathology leading to increased fracture-risk. MicroRNAs (miRNAs) are the most abundant RNA species to be found in cell-free blood. Encapsulated within microvesicles or bound to proteins, circulating miRNAs are remarkably stable analytes that can be measured using gold-standard technologies such as quantitative polymerase-chain-reaction (qPCR). Nevertheless, the analysis of circulating miRNAs faces several pre-analytical as well as analytical challenges. From a biological view, there is accumulating evidence that miRNAs play essential roles in the regulation of various biological processes including bone homeostasis. Moreover, specific changes in miRNA transcription levels or miRNA secretory levels have been linked to the development and progression of certain bone diseases. Only recently, results from circulating miRNAs analysis in patients with osteopenia, osteoporosis and fragility fractures have been reported. By comparing these findings to studies on circulating miRNAs in cellular senescence and aging or muscle physiology and sarcopenia, several overlaps were observed. This suggests that signatures observed during osteoporosis might not be specific to the pathophysiology in bone, but rather integrate information from several tissue types. Despite these promising first data, more work remains to be done until circulating miRNAs can serve as established and robust diagnostic tools for bone diseases in clinical research, clinical routine and in personalized medicine. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Circulating miR-1, miR-133a, and miR-206 levels are increased after a half-marathon run.

PubMed

Gomes, Clarissa P C; Oliveira, Getúlio P; Madrid, Bibiano; Almeida, Jeeser A; Franco, Octávio L; Pereira, Rinaldo W

2014-11-01

Circulating miRNAs are potential biomarkers that can be important molecules driving cell-to-cell communication. To investigate circulating muscle-specific miRNAs in recreational athletes. Three miRNAs from whole plasma before and after a half-marathon were analyzed by qPCR. MiR-1, -133a, and -206 significantly increased after the race. Increased levels of miRNAs after exercise point to potential biomarkers and to the possibility of being functional players following endurance training. These miRNAs are potential biomarkers of muscle damage or adaptation to exercise.

Differences in microRNA expression during tumor development in the transition and peripheral zones of the prostate

PubMed Central

2013-01-01

Background The prostate is divided into three glandular zones, the peripheral zone (PZ), the transition zone (TZ), and the central zone. Most prostate tumors arise in the peripheral zone (70-75%) and in the transition zone (20-25%) while only 10% arise in the central zone. The aim of this study was to investigate if differences in miRNA expression could be a possible explanation for the difference in propensity of tumors in the zones of the prostate. Methods Patients with prostate cancer were included in the study if they had a tumor with Gleason grade 3 in the PZ, the TZ, or both (n=16). Normal prostate tissue was collected from men undergoing cystoprostatectomy (n=20). The expression of 667 unique miRNAs was investigated using TaqMan low density arrays for miRNAs. Student’s t-test was used in order to identify differentially expressed miRNAs, followed by hierarchical clustering and principal component analysis (PCA) to study the separation of the tissues. The ADtree algorithm was used to identify markers for classification of tissues and a cross-validation procedure was used to test the generality of the identified miRNA-based classifiers. Results The t-tests revealed that the major differences in miRNA expression are found between normal and malignant tissues. Hierarchical clustering and PCA based on differentially expressed miRNAs between normal and malignant tissues showed perfect separation between samples, while the corresponding analyses based on differentially expressed miRNAs between the two zones showed several misplaced samples. A classification and cross-validation procedure confirmed these results and several potential miRNA markers were identified. Conclusions The results of this study indicate that the major differences in the transcription program are those arising during tumor development, rather than during normal tissue development. In addition, tumors arising in the TZ have more unique differentially expressed miRNAs compared to the PZ. The results also indicate that separate miRNA expression signatures for diagnosis might be needed for tumors arising in the different zones. MicroRNA signatures that are specific for PZ and TZ tumors could also lead to more accurate prognoses, since tumors arising in the PZ tend to be more aggressive than tumors arising in the TZ. PMID:23890084

Circular RNA: A new star of noncoding RNAs.

PubMed

Qu, Shibin; Yang, Xisheng; Li, Xiaolei; Wang, Jianlin; Gao, Yuan; Shang, Runze; Sun, Wei; Dou, Kefeng; Li, Haimin

2015-09-01

Circular RNAs (circRNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. Recent studies have discovered thousands of endogenous circRNAs in mammalian cells. CircRNAs are largely generated from exonic or intronic sequences, and reverse complementary sequences or RNA-binding proteins (RBPs) are necessary for circRNA biogenesis. The majority of circRNAs are conserved across species, are stable and resistant to RNase R, and often exhibit tissue/developmental-stage-specific expression. Recent research has revealed that circRNAs can function as microRNA (miRNA) sponges, regulators of splicing and transcription, and modifiers of parental gene expression. Emerging evidence indicates that circRNAs might play important roles in atherosclerotic vascular disease risk, neurological disorders, prion diseases and cancer; exhibit aberrant expression in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC); and serve as diagnostic or predictive biomarkers of some diseases. Similar to miRNAs and long noncoding RNAs (lncRNAs), circRNAs are becoming a new research hotspot in the field of RNA and could be widely involved in the processes of life. Herein, we review the formation and properties of circRNAs, their functions, and their potential significance in disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

PubMed

Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

2017-02-01

MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

Signaling Pathways in Pathogenesis of Diamond Blackfan Anemia

DTIC Science & Technology

2015-12-01

abnormalities, and predisposition to cancer. The current treatment of steroids and chronic transfusions leads to significant morbidity. Approximately 25...more rigorously test this hypothesis and identify new downstream targets and microRNAs, we propose three specific aims. In Aim 1, we will...aplasia, congenital abnormalities, and predisposition to cancer. The current treatment of steroids and chronic transfusions leads to significant

Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.

PubMed

Suárez, Yajaira; Fernández-Hernando, Carlos; Yu, Jun; Gerber, Scott A; Harrison, Kenneth D; Pober, Jordan S; Iruela-Arispe, M Luisa; Merkenschlager, Matthias; Sessa, William C

2008-09-16

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the up-regulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.

Emerging strategies for RNA interference (RNAi) applications in insects.

PubMed

Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

2015-01-01

RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

PubMed

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J; Jopling, Catherine L

2015-04-01

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

Apoptosis of Limb Innervating Motor Neurons and Erosion of Motor Pool Identity Upon Lineage Specific Dicer Inactivation

PubMed Central

Chen, Jun-An; Wichterle, Hynek

2012-01-01

Diversification of mammalian spinal motor neurons into hundreds of subtypes is critical for the maintenance of body posture and coordination of complex movements. Motor neuron differentiation is controlled by extrinsic signals that regulate intrinsic genetic programs specifying and consolidating motor neuron subtype identity. While transcription factors have been recognized as principal regulators of the intrinsic program, the role of posttranscriptional regulations has not been systematically tested. MicroRNAs produced by Dicer mediated cleavage of RNA hairpins contribute to gene regulation by posttranscriptional silencing. Here we used Olig2-cre conditional deletion of Dicer gene in motor neuron progenitors to examine effects of miRNA biogenesis disruption on postmitotic spinal motor neurons. We report that despite the initial increase in the number of motor neuron progenitors, disruption of Dicer function results in a loss of many limb- and sympathetic ganglia-innervating spinal motor neurons. Furthermore, it leads to defects in motor pool identity specification. Thus, our results indicate that miRNAs are an integral part of the genetic program controlling motor neuron survival and acquisition of subtype specific properties. PMID:22629237

Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics

PubMed Central

2012-01-01

Background MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. Results A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Conclusions Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress. PMID:22455456

MicroRNAs (miRNAs) as biomarker(s) for prognosis and diagnosis of gastrointestinal (GI) cancers.

PubMed

Macha, Muzafar A; Seshacharyulu, Parthasarathy; Krishn, Shiv Ram; Pai, Priya; Rachagani, Satyanarayana; Jain, Maneesh; Batra, Surinder K

2014-01-01

Gastrointestinal (GI) cancers remain one of the most common malignancies and are the second common cause of cancer deaths worldwide. The limited effectiveness of therapy for patients with advanced stage and recurrent disease is a reflection of an incomplete understanding of the molecular basis of GI carcinogenesis. Major advancements have improved our understanding of pathology and pathogenesis of GI cancers, but high mortality rates, unfavorable prognosis and lack of clinical predictive biomarkers provide an impetus to investigate new sensitive and specific diagnostic and prognostic markers for GI cancers. MicroRNAs (miRNAs) are short (19-24 nucleotides) noncoding RNA molecules that regulate gene expression at the posttranscriptional level thus playing an important role in modulating various biological processes including, but not limited to developmental processes, proliferation, apoptosis, metabolism, differentiation, epithelial-mechenchymal transition and are involved in the initiation and progression of various human cancers. Unique miRNA expression profiles have been observed in various cancer types at different stages, suggesting their potential as diagnostic and prognostic biomarkers. Due to their tumor-specific and tissue-specific expression profiles, stability, robust clinical assays for detection in serum as well as in formalin-fixed tissue samples, miRNAs have emerged as attractive candidates for diagnostic and prognostic applications. This review summarizes recent research supporting the utility of miRNAs as novel diagnostic and prognostic tools for GI cancers.

MicroRNAs (miRNAs) as Biomarker(s) for Prognosis and Diagnosis of Gastrointestinal (GI) Cancers

PubMed Central

Macha, Muzafar A.; Seshacharyulu, Parthasarathy; Krishn, Shiv Ram; Pai, Priya; Rachagani, Satyanarayana; Jain, Maneesh; Batra, Surinder K.

2014-01-01

Gastrointestinal (GI) cancers remain one of the most common malignancies and are the second common cause of cancer deaths worldwide. The limited effectiveness of therapy for patients with advanced stage and recurrent disease is a reflection of an incomplete understanding of the molecular basis of GI carcinogenesis. Major advancements have improved our understanding of pathology and pathogenesis of GI cancers, but high mortality rates, unfavorable prognosis and lack of clinical predictive biomarkers provide an impetus to investigate new sensitive and specific diagnostic and prognostic markers for GI cancers. MicroRNAs (miRNAs) are short (19–24 nucleotides) noncoding RNA molecules that regulate gene expression at the posttranscriptional level thus playing an important role in modulating various biological processes including, but not limited, to developmental processes, proliferation, apoptosis, metabolism, differentiation, epithelial-mechenchymal transition and are involved in the initiation and progression of various human cancers. Unique miRNA expression profiles have been observed in various cancer types at different stages, suggesting their potential as diagnostic and prognostic biomarkers. Due to their tumor-specific and tissue-specific expression profiles, stability, robust clinical assays for detection in serum as well as in formalin-fixed tissue samples, miRNAs have emerged as attractive candidates for diagnostic and prognostic applications. This review summarizes recent research supporting the utility of miRNAs as novel diagnostic and prognostic tools for GI cancers. PMID:24479799

Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa)

PubMed Central

Ding, Yanfei; Chen, Zhen; Zhu, Cheng

2011-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate specific target mRNAs at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptations to environmental stresses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants. To investigate the responsive functions of miRNAs under Cd stress, miRNA expression in Cd-stressed rice (Oryza sativa) was profiled using a microarray assay. A total of 19 Cd-responsive miRNAs were identified, of which six were further validated experimentally. Target genes were also predicted for these Cd-responsive miRNAs, which encoded transcription factors, and proteins associated with metabolic processes or stress responses. In addition, the mRNA levels of several targets were negatively correlated with the corresponding miRNAs under Cd stress. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a novel molecular mechanism of heavy metal tolerance in plants. PMID:21362738

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles

PubMed Central

Caswell, Clayton C.; Oglesby-Sherrouse, Amanda G.; Murphy, Erin R.

2014-01-01

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators. PMID:25389522

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

PubMed

Caswell, Clayton C; Oglesby-Sherrouse, Amanda G; Murphy, Erin R

2014-01-01

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

PubMed

Suzuki, Atsushi; Niimi, Yuki; Shinmyozu, Kaori; Zhou, Zhi; Kiso, Makoto; Saga, Yumiko

2016-01-01

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development. © 2015 The Authors.

CstF-64 and 3'-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα.

PubMed

Kandala, Divya T; Mohan, Nimmy; A, Vivekanand; A P, Sudheesh; G, Reshmi; Laishram, Rakesh S

2016-01-29

Almost all eukaryotic mRNAs have a poly (A) tail at the 3'-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3'-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3'-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3'-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

PubMed Central

Kandala, Divya T.; Mohan, Nimmy; A, Vivekanand; AP, Sudheesh; G, Reshmi; Laishram, Rakesh S.

2016-01-01

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation. PMID:26496945

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

PubMed Central

Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

Short interfering RNA confers intracellular antiviral immunity in human cells.

PubMed

Gitlin, Leonid; Karelsky, Sveta; Andino, Raul

2002-07-25

Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. In plants, it serves as an antiviral defence mechanism. Animal cells also possess this machinery but its specific function is unclear. Here we demonstrate that dsRNA can effectively protect human cells against infection by a rapidly replicating and highly cytolytic RNA virus. Pre-treatment of human and mouse cells with double-stranded, short interfering RNAs (siRNAs) to the poliovirus genome markedly reduces the titre of virus progeny and promotes clearance of the virus from most of the infected cells. The antiviral effect is sequence-specific and is not attributable to either classical antisense mechanisms or to interferon and the interferon response effectors protein kinase R (PKR) and RNaseL. Protection is the result of direct targeting of the viral genome by siRNA, as sequence analysis of escape virus (resistant to siRNAs) reveals one nucleotide substitution in the middle of the targeted sequence. Thus, siRNAs elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses.

Real-time functional imaging for monitoring miR-133 during myogenic differentiation.

PubMed

Kato, Yoshio; Miyaki, Shigeru; Yokoyama, Shigetoshi; Omori, Shin; Inoue, Atsushi; Horiuchi, Machiko; Asahara, Hiroshi

2009-11-01

MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.

Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs from roots of Medicago sativa and Medicago truncatula.

PubMed

Long, Rui-Cai; Li, Ming-Na; Kang, Jun-Mei; Zhang, Tie-Jun; Sun, Yan; Yang, Qing-Chuan

2015-05-01

Small 21- to 24-nucleotide (nt) ribonucleic acids (RNAs), notably the microRNA (miRNA), are emerging as a posttranscriptional regulation mechanism. Salt stress is one of the primary abiotic stresses that cause the crop losses worldwide. In saline lands, root growth and function of plant are determined by the action of environmental salt stress through specific genes that adapt root development to the restrictive condition. To elucidate the role of miRNAs in salt stress regulation in Medicago, we used a high-throughput sequencing approach to analyze four small RNA libraries from roots of Zhongmu-1 (Medicago sativa) and Jemalong A17 (Medicago truncatula), which were treated with 300 mM NaCl for 0 and 8 h. Each library generated about 20 million short sequences and contained predominantly small RNAs of 24-nt length, followed by 21-nt and 22-nt small RNAs. Using sequence analysis, we identified 385 conserved miRNAs from 96 families, along with 68 novel candidate miRNAs. Of all the 68 predicted novel miRNAs, 15 miRNAs were identified to have miRNA*. Statistical analysis on abundance of sequencing read revealed specific miRNA showing contrasting expression patterns between M. sativa and M. truncatula roots, as well as between roots treated for 0 and 8 h. The expression of 10 conserved and novel miRNAs was also quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The miRNA precursor and target genes were predicted by bioinformatics analysis. We concluded that the salt stress related conserved and novel miRNAs may have a large variety of target mRNAs, some of which might play key roles in salt stress regulation of Medicago. © 2014 Scandinavian Plant Physiology Society.

Mammalian genome-wide loss-of-function screens using arrayed small interfering RNA expression libraries.

PubMed

Zheng, Lianxing; Ding, Sheng

2004-04-01

Extract: RNA interference (RNAi), first discovered in Caenorhabdtitis elegans and now widely found and applied in a variety of organisms such as Drosophila, zebrafish and mammalian systems, has emerged to revolutionize the field of functional genomics by inducing specific and effective post-transcriptional gene silencing for loss-of-function studies. Mechanistic investigations of RNAi suggest that long double-stranded RNAs (dsRNAs) are first cleaved by the RNase III-like enzyme, Dicer, to 21-23 base pair (bp) small interfering RNAs (siRNAs). These siRNAs are resolved by ATP-dependent RNA helicase, and the resulting single-stranded RNAs are then incorporated into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex guides the RISC to the homologous mRNA, where the RISC-associated endoribonuclease cleaves the target mRNA, resulting in silencing of the target gene. The approach of using long dsRNA (up to 1-2 kb) in C. elegans and Drosophila to induce gene silencing cannot be similarly used in mammalian cells, where introduction of long dsRNA activates the dsRNA-dependent protein kinase PKR. PKR phosphorylates and inactivates the translation initiation factor eIF2, resulting in a non-specific gene-silencing effect. Development and implementation of the use of 21 to 23bp siRNAs, which can be prepared by chemical synthesis, in vitro transcription, or expressed in cells using siRNA expression systems, allows specific and effective gene silencing in mammalian cells to occur without activation of PKR.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.

PubMed

James, Amanda Marie; Baker, Meredith B; Bao, Gang; Searles, Charles D

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro . The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

PubMed Central

James, Amanda Marie; Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples. PMID:28255356

MicroRNAs and lipoproteins: a connection beyond atherosclerosis?

PubMed Central

Norata, Giuseppe Danilo; Sala, Federica; Catapano, Alberico Luigi; Fernández-Hernando, Carlos

2014-01-01

MicroRNAs (miRNAs) are involved in the pathogenesis of a number of cardiovascular diseases. In this review article, we have summarized the role of miRNAs in regulating lipid metabolism and how their therapeutical inhibition may lead to new approaches to treat cardiometabolic diseases, including atherosclerosis and metabolic syndrome. Specific miRNAs, such as miR-33a and -33b, represent one of the most interesting and attractive targets for metabolic-related disorders and anti-miR33 approaches are under intensive investigation. In addition to miR-33, other miRNAs, including miR-122, are also emerging as key players in lipid metabolism. More recently miRNAs were shown to exert their activities in a paracrine manner and also systemically. The latter is possible due to lipid-carriers, including lipoproteins, that transport and protect miRNAs from degradation. The emerging strong connection between miRNAs, lipoproteins and lipid metabolism indicates the existence of a reciprocal modulation that might go beyond atherosclerosis. PMID:23260873

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

PubMed Central

Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

2007-01-01

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

Transcription start site associated RNAs (TSSaRNAs) are ubiquitous in all domains of life.

PubMed

Zaramela, Livia S; Vêncio, Ricardo Z N; ten-Caten, Felipe; Baliga, Nitin S; Koide, Tie

2014-01-01

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes.

PubMed

Zhao, Ying-Tao; Wang, Meng; Fu, San-Xiong; Yang, Wei-Cai; Qi, Cun-Kou; Wang, Xiu-Jie

2012-02-01

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

Decoding the Emerging Patterns Exhibited in Non-coding RNAs Characteristic of Lung Cancer with Regard to their Clinical Significance.

PubMed

Sonea, Laura; Buse, Mihail; Gulei, Diana; Onaciu, Anca; Simon, Ioan; Braicu, Cornelia; Berindan-Neagoe, Ioana

2018-05-01

Lung cancer continues to be the leading topic concerning global mortality rate caused by can-cer; it needs to be further investigated to reduce these dramatic unfavorable statistic data. Non-coding RNAs (ncRNAs) have been shown to be important cellular regulatory factors and the alteration of their expression levels has become correlated to extensive number of pathologies. Specifically, their expres-sion profiles are correlated with development and progression of lung cancer, generating great interest for further investigation. This review focuses on the complex role of non-coding RNAs, namely miR-NAs, piwi-interacting RNAs, small nucleolar RNAs, long non-coding RNAs and circular RNAs in the process of developing novel biomarkers for diagnostic and prognostic factors that can then be utilized for personalized therapies toward this devastating disease. To support the concept of personalized medi-cine, we will focus on the roles of miRNAs in lung cancer tumorigenesis, their use as diagnostic and prognostic biomarkers and their application for patient therapy.

Use of biomarkers to establish potential role and function of circulating microRNAs in acute heart failure.

PubMed

Vegter, Eline L; Schmitter, Daniela; Hagemeijer, Yanick; Ovchinnikova, Ekaterina S; van der Harst, Pim; Teerlink, John R; O'Connor, Christopher M; Metra, Marco; Davison, Beth A; Bloomfield, Daniel; Cotter, Gad; Cleland, John G; Givertz, Michael M; Ponikowski, Piotr; van Veldhuisen, Dirk J; van der Meer, Peter; Berezikov, Eugene; Voors, Adriaan A; Khan, Mohsin A F

2016-12-01

Circulating microRNAs (miRNAs) emerge as potential heart failure biomarkers. We aimed to identify associations between acute heart failure (AHF)-specific circulating miRNAs and well-known heart failure biomarkers. Associations between 16 biomarkers predictive for 180day mortality and the levels of 12 AHF-specific miRNAs were determined in 100 hospitalized AHF patients, at baseline and 48hours. Patients were divided in 4 pre-defined groups, based on clinical parameters during hospitalization. Correlation analyses between miRNAs and biomarkers were performed and complemented by miRNA target prediction and pathway analysis. No significant correlations were found at hospital admission. However, after 48hours, 7 miRNAs were significantly negatively correlated to biomarkers indicative for a worse clinical outcome in the patient group with the most unfavorable in-hospital course (n=21); miR-16-5p was correlated to C-reactive protein (R=-0.66, p-value=0.0027), miR-106a-5p to creatinine (R=-0.68, p-value=0.002), miR-223-3p to growth differentiation factor 15 (R=-0.69, p-value=0.0015), miR-652-3p to soluble ST-2 (R=-0.77, p-value<0.001), miR-199a-3p to procalcitonin (R=-0.72, p-value<0.001) and galectin-3 (R=-0.73, p-value<0.001) and miR-18a-5p to procalcitonin (R=-0.68, p-value=0.002). MiRNA target prediction and pathway analysis identified several pathways related to cardiac diseases, which could be linked to some of the miRNA-biomarker correlations. The majority of correlations between circulating AHF-specific miRNAs were related to biomarkers predictive for a worse clinical outcome in a subgroup of worsening heart failure patients at 48hours of hospitalization. The selective findings suggest a time-dependent effect of circulating miRNAs and highlight the susceptibility to individual patient characteristics influencing potential relations between miRNAs and biomarkers. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

PubMed Central

Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

2016-01-01

Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

Are microRNAs true sensors of ageing and cellular senescence?

PubMed

Williams, Justin; Smith, Flint; Kumar, Subodh; Vijayan, Murali; Reddy, P Hemachandra

2017-05-01

All living beings are programmed to death due to aging and age-related processes. Aging is a normal process of every living species. While all cells are inevitably progressing towards death, many disease processes accelerate the aging process, leading to senescence. Pathologies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cardiovascular disease, cancer, and skin diseases have been associated with deregulated aging. Healthy aging can delay onset of all age-related diseases. Genetics and epigenetics are reported to play large roles in accelerating and/or delaying the onset of age-related diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent molecular biology discoveries have revealed that microRNAs (miRNAs) are potential sensors of aging and cellular senescence. Due to miRNAs capability to bind to the 3' untranslated region (UTR) of mRNA of specific genes, miRNAs can prevent the translation of specific genes. The purpose of our article is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging and senescence. Our article discusses the current understanding of cellular senescence, its interplay with miRNAs regulation, and how they both contribute to disease processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

Computational analysis of microRNA function in heart development.

PubMed

Liu, Ganqiang; Ding, Min; Chen, Jiajia; Huang, Jinyan; Wang, Haiyun; Jing, Qing; Shen, Bairong

2010-09-01

Emerging evidence suggests that specific spatio-temporal microRNA (miRNA) expression is required for heart development. In recent years, hundreds of miRNAs have been discovered. In contrast, functional annotations are available only for a very small fraction of these regulatory molecules. In order to provide a global perspective for the biologists who study the relationship between differentially expressed miRNAs and heart development, we employed computational analysis to uncover the specific cellular processes and biological pathways targeted by miRNAs in mouse heart development. Here, we utilized Gene Ontology (GO) categories, KEGG Pathway, and GeneGo Pathway Maps as a gene functional annotation system for miRNA target enrichment analysis. The target genes of miRNAs were found to be enriched in functional categories and pathway maps in which miRNAs could play important roles during heart development. Meanwhile, we developed miRHrt (http://sysbio.suda.edu.cn/mirhrt/), a database aiming to provide a comprehensive resource of miRNA function in regulating heart development. These computational analysis results effectively illustrated the correlation of differentially expressed miRNAs with cellular functions and heart development. We hope that the identified novel heart development-associated pathways and the database presented here would facilitate further understanding of the roles and mechanisms of miRNAs in heart development.

miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects.

PubMed

Kappel, Andreas; Keller, Andreas

2017-05-01

microRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression in eukaryotes. Their differential abundance is indicative or even causative for a variety of pathological processes including cancer or cardiovascular disorders. Due to their important biological function, miRNAs represent a promising class of novel biomarkers that may be used to diagnose life-threatening diseases, and to monitor disease progression. Further, they may guide treatment selection or dosage of drugs. miRNAs from blood or derived fractions are particularly interesting candidates for routine laboratory applications, as they can be measured in most clinical laboratories already today. This assures a good accessibility of respective tests. Albeit their great potential, miRNA-based diagnostic tests have not made their way yet into the clinical routine, and hence no standardized workflows have been established to measure miRNAs for patients' benefit. In this review we summarize the detection technologies and workflow options that exist to measure miRNAs, and we describe the advantages and disadvantages of each of these options. Moreover, we also provide a perspective on data analysis aspects that are vital for translation of raw data into actionable diagnostic test results.

Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis

USDA-ARS?s Scientific Manuscript database

/Cis/-nat-siRNAs are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic /cis/-nat-siRNAs that has been described to date is in envir...

Plant-based microRNA presences in mice and human sera to breast milk

USDA-ARS?s Scientific Manuscript database

Plant foods contain hundreds of thousands of different small RNAs, including microRNAs (miRNAs). A microRNA (miRNA) is a tiny (19-24 nucleotide) piece of RNA that attaches to a specific protein-making mRNA, thus inhibiting protein production. A recent finding shows that a miRNA in rice survives dige...

Identification of circular RNA in the Bombyx mori silk gland.

PubMed

Gan, Huaiyan; Feng, Tieshan; Wu, Yuqian; Liu, Chun; Xia, Qingyou; Cheng, Tingcai

2017-10-01

Bombyx mori is an economically important holometabolous lepidopteran insect. In B. mori endogenous noncoding RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs play crucial biological functions in metamorphosis and sex determination. In addition, circular RNAs (circRNAs) have been recently identified as noncoding RNAs in most common model organisms and show potential as gene regulators. However, to date, there have been few studies on the circRNAs present in the B. mori genome conducted to date. Here, we identified 3916 circRNAs by deep circular transcriptome sequencing using the silk gland of B. mori. 3155 circRNAs were found to be derived from 1727 parental genes. The circRNAs displayed tissue-specific expression between the middle silk gland (MSG) and posterior silk gland (PSG), with 2532 and 880 being upregulated circRNAs in the MSG and PSG, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parental genes from the MSG and PSG were generally annotated to similar categories and pathways. The interaction network of circRNAs and miRNAs showed that circRNAs might act as miRNA sponges or interact with miRNAs in some other way. Overall, the results revealed the complicated patterns of circRNAs in the B. mori silk gland providing a new angle from which to explore the mechanisms of complex gene regulation and efficient silk protein synthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

PubMed Central

Ge, Xie; Zhang, Yong; Jiang, Jianhao; Zhong, Yi; Yang, Xiaonan; Li, Zhiqian; Huang, Yongping; Tan, Anjiang

2013-01-01

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs. PMID:23289012

Effector and regulatory dendritic cells display distinct patterns of miRNA expression.

PubMed

Lombardi, Vincent; Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet-Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron-Bodo, Véronique; Moingeon, Philippe

2017-09-01

MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Using specific culture conditions, we differentiated immature human monocyte-derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of T H 1, T H 2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non-allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR-132 and miR-155), was down-regulated compared to non-allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow-up AIT efficacy. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression

PubMed Central

Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet‐Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron‐Bodo, Véronique; Moingeon, Philippe

2017-01-01

Abstract Introduction MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Method Using specific culture conditions, we differentiated immature human monocyte‐derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of TH1, TH2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non‐allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. Results We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR‐132 and miR‐155), was down‐regulated compared to non‐allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Conclusions Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow‐up AIT efficacy. PMID:28497578

Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

PubMed

Izuogu, Osagie G; Alhasan, Abd A; Mellough, Carla; Collin, Joseph; Gallon, Richard; Hyslop, Jonathon; Mastrorosa, Francesco K; Ehrmann, Ingrid; Lako, Majlinda; Elliott, David J; Santibanez-Koref, Mauro; Jackson, Michael S

2018-04-20

Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

Small RNA Analysis in Sindbis Virus Infected Human HEK293 Cells

PubMed Central

Dalmay, Tamas; Powell, Penny P.

2013-01-01

Introduction In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells. Results SINV AR339 and TR339-GFP were adapted to grow in HEK293 cells. Deep sequencing of small RNAs (sRNAs) early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral sRNAs (vsRNAs), with no size, sequence or location specific patterns characteristic of Dicer products nor did they possess any discernible pattern to ascribe to a specific RNAi biogenesis pathway. This was supported by multiple variants for each sequence, and lack of hot spots along the viral genome sequence. The abundance of the best defined vsRNAs was below the limit of Northern blot detection. The adaptation of the virus to HEK293 cells showed little sequence changes compared to the reference; however, a SNP in E1 gene with a preference from G to C was found. Deep sequencing results showed little variation of expression of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed no difference in expression by Northern blot analysis. Conclusions We show that, unlike SINV infection of invertebrates, generation of Dicer-dependent svRNAs and change in expression of cellular miRNAs were not detected as part of the Human response to SINV. PMID:24391886

Birth, coming of age and death: The intriguing life of long noncoding RNAs.

PubMed

Samudyata; Castelo-Branco, Gonçalo; Bonetti, Alessandro

2018-07-01

Mammalian genomes are pervasively transcribed, with long noncoding RNAs being the most abundant fraction. Recent studies have highlighted the central role played by these transcripts in several physiological and pathological processes. Despite several metabolic features shared between coding and noncoding transcripts, these two classes of RNAs exhibit multiple differences regarding their biogenesis and processing. Here we review such distinctions, focusing on the unique features of specific long noncoding RNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sex hormones regulate cerebral drug metabolism via brain miRNAs: down-regulation of brain CYP2D by androgens reduces the analgesic effects of tramadol

PubMed Central

Li, Jie; Xie, Mengmeng; Wang, Xiaoshuang; Ouyang, Xiufang; Wan, Yu; Dong, Guicheng; Yang, Zheqiong; Yang, Jing; Yue, Jiang

2015-01-01

Background and Purpose Brain cytochrome P450 2D (CYP2D) metabolises exogenous neurotoxins, endogenous substances and neurotransmitters. Brain CYP2D can be regulated in an organ-specific manner, but the possible regulatory mechanisms are poorly understood. We investigated the involvement of miRNAs in the selective regulation of brain CYP2D by testosterone and the corresponding alteration of the pharmacological profiles of tramadol by testosterone. Experimental Approach The regulation of CYP2D and brain-enriched miRNAs by testosterone was investigated using SH-SY5Y cells, U251 cells, and HepG2 cells as well as orchiectomized growth hormone receptor knockout (GHR-KO) mice and rats. Concentration–time curves of tramadol in rat brain were determined using a microdialysis technique. The analgesic action of tramadol was assessed by the tail-flick test in rats. Key Results miR-101 and miR-128-2 bound the 3′-untranslated region of the CYP2D6 mRNA and decreased its level. Testosterone decreased CYP2D6 catalytic function via the up-regulation of miR-101 and miR-128-2 in SH-SY5Y and U251 cells, but not in HepG2 cells. Orchiectomy decreased the levels of miR-101 and miR-128-2 in the hippocampus of male GHR-KO mice, indicating that androgens regulate miRNAs directly, not via the alteration of growth hormone secretion patterns. Changes in the pharmacokinetic and pharmacodynamic profiles of tramadol by orchiectomy was attenuated by either testosterone supplementation or a specific brain CYP2D inhibitor. Conclusions and Implications The selective regulation of brain CYP2D via brain-enriched miRNAs, following changes in androgen levels, such as in testosterone therapy, androgen deprivation therapy and/or ageing may alter the response to centrally active substances. PMID:26031356

Bioinformatics Analysis of Small RNAs in Pima (Gossypium barbadense L.)

PubMed Central

Hu, Hongtao; Yu, Dazhao; Liu, Hong

2015-01-01

Small RNAs (sRNAs) are ~20 to 24 nucleotide single-stranded RNAs that play crucial roles in regulation of gene expression. In plants, sRNAs are classified into microRNAs (miRNAs), repeat-associated siRNAs (ra-siRNAs), phased siRNAs (pha-siRNAs), cis and trans natural antisense transcript siRNAs (cis- and trans-nat siRNAs). Pima (Gossypium barbadense L.) is one of the most economically important fiber crops, producing the best and longest spinnable fiber. Although some miRNAs are profiled in Pima, little is known about siRNAs, the largest subclass of plant sRNAs. In order to profile these gene regulators in Pima, a comprehensive analysis of sRNAs was conducted by mining publicly available sRNA data, leading to identification of 678 miRNAs, 3,559,126 ra-siRNAs, 627 pha-siRNAs, 136,600 cis-nat siRNAs and 79,994 trans-nat siRNAs. The 678 miRNAs, belonging to 98 conserved and 402 lineage-specific families, were produced from 2,138 precursors, of which 297 arose from introns, exons, or intron/UTR-exon junctions of protein-coding genes. Ra-siRNAs were produced from various repeat loci, while most (97%) were yielded from retrotransposons, especially LTRs (long terminal repeats). The genes encoding auxin-signaling-related proteins, NBS-LRRs and transcription factors were major sources of pha-siRNAs, while two conserved TAS3 homologs were found as well. Most cis-NATs in Pima overlapped in enclosed and convergent orientations, while a few hybridized in divergent and coincided orientations. Most cis- and trans-nat siRNAs were produced from overlapping regions. Additionally, characteristics of length and the 5’-first nucleotide of each sRNA class were analyzed as well. Results in this study created a valuable molecular resource that would facilitate studies on mechanism of controlling gene expression. PMID:25679373

Systematic identification and comparison of expressed profiles of lncRNAs and circRNAs with associated co-expression and ceRNA networks in mouse germline stem cells

PubMed Central

Wu, Ji

2017-01-01

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) involve in germ cell development. However, little is known about the functions and mechanisms of lncRNAs and circRNAs in self-renewal and differentiation of germline stem cells. Therefore, we explored the expression profiles of mRNAs, lncRNAs, and circRNAs in male and female mouse germline stem cells by high-throughput sequencing. We identified 18573 novel lncRNAs and 18822 circRNAs in the germline stem cells and further confirmed the existence of these lncRNAs and circRNAs by RT-PCR. The results showed that male and female germline stem cells had similar GDNF signaling mechanism. Subsequently, 8115 mRNAs, 3996 lncRNAs, and 921 circRNAs exhibited sex-biased expression that may be associated with germline stem cell acquisition of the sex-specific properties required for differentiation into gametes. Gene Ontology (GO) and KEGG pathway enrichment analyses revealed different functions for these sex-biased lncRNAs and circRNAs. We further constructed correlated expression networks including coding–noncoding co-expression and competing endogenous RNAs with bioinformatics. Co-expression analysis showed hundreds of lncRNAs were correlated with sex differences in mouse germline stem cells, including lncRNA Gm11851, lncRNA Gm12840, lncRNA 4930405O22Rik, and lncRNA Atp10d. CeRNA network inferred that lncRNA Meg3 and cirRNA Igf1r could bind competitively with miRNA-15a-5p increasing target gene Inha, Acsl3, Kif21b, and Igfbp2 expressions. These findings provide novel perspectives on lncRNAs and circRNAs and lay a foundation for future research into the regulating mechanisms of lncRNAs and circRNAs in germline stem cells. PMID:28404936

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

PubMed

Liu, Jun-Ying; Fan, Hui-Yan; Wang, Ying; Zhang, Yong-Liang; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

The circulating non-coding RNA landscape for biomarker research: lessons and prospects from cardiovascular diseases.

PubMed

St Ecedil Pień, Ewa; Costa, Marina C; Kurc, Szczepan; Drożdż, Anna; Cortez-Dias, Nuno; Enguita, Francisco J

2018-06-07

Pervasive transcription of the human genome is responsible for the production of a myriad of non-coding RNA molecules (ncRNAs) some of them with regulatory functions. The pivotal role of ncRNAs in cardiovascular biology has been unveiled in the last decade, starting from the characterization of the involvement of micro-RNAs in cardiovascular development and function, and followed by the use of circulating ncRNAs as biomarkers of cardiovascular diseases. The human non-coding secretome is composed by several RNA species that circulate in body fluids and could be used as biomarkers for diagnosis and outcome prediction. In cardiovascular diseases, secreted ncRNAs have been described as biomarkers of several conditions including myocardial infarction, cardiac failure, and atrial fibrillation. Among circulating ncRNAs, micro-RNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have been proposed as biomarkers in different cardiovascular diseases. In comparison with standard biomarkers, the biochemical nature of ncRNAs offers better stability and flexible storage conditions of the samples, and increased sensitivity and specificity. In this review we describe the current trends and future prospects of the use of the ncRNA secretome components as biomarkers of cardiovascular diseases, including the opening questions related with their secretion mechanisms and regulatory actions.

Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery.

PubMed

Donovan, Jesse; Rath, Sneha; Kolet-Mandrikov, David; Korennykh, Alexei

2017-11-01

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest. © 2017 Donovan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Sources and functions of extracellular small RNAs in human circulation

PubMed Central

Fritz, Joëlle V.; Heintz-Buschart, Anna; Ghosal, Anubrata; Wampach, Linda; Etheridge, Alton; Galas, David; Wilmes, Paul

2017-01-01

Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation including microRNAs, tRNA, rRNA and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease contexts and have therefore been proposed as well-suited biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in receiving cells. Albeit controversial, exogenous ex-sRNAs derived from plants and microorganisms have also been described in human blood. Essential questions which remain to be conclusively addressed in the field concern the (i) presence and mechanistic sources of exogenous ex-sRNA in human bodily fluids, (ii) detection and measurement of ex-sRNA in human circulation, (iii) selectivity of ex-sRNA export and import, (iv) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (v) cell-, tissue-, organ- and organism-wide impacts of ex-sRNAs. We will survey the present state of knowledge of most of these questions in this review. PMID:27215587

Salivary miRNA profiles identify children with autism spectrum disorder, correlate with adaptive behavior, and implicate ASD candidate genes involved in neurodevelopment.

PubMed

Hicks, Steven D; Ignacio, Cherry; Gentile, Karen; Middleton, Frank A

2016-04-22

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder that lacks adequate screening tools, often delaying diagnosis and therapeutic interventions. Despite a substantial genetic component, no single gene variant accounts for >1 % of ASD incidence. Epigenetic mechanisms that include microRNAs (miRNAs) may contribute to the ASD phenotype by altering networks of neurodevelopmental genes. The extracellular availability of miRNAs allows for painless, noninvasive collection from biofluids. In this study, we investigated the potential for saliva-based miRNAs to serve as diagnostic screening tools and evaluated their potential functional importance. Salivary miRNA was purified from 24 ASD subjects and 21 age- and gender-matched control subjects. The ASD group included individuals with mild ASD (DSM-5 criteria and Autism Diagnostic Observation Schedule) and no history of neurologic disorder, pre-term birth, or known chromosomal abnormality. All subjects completed a thorough neurodevelopmental assessment with the Vineland Adaptive Behavior Scales at the time of saliva collection. A total of 246 miRNAs were detected and quantified in at least half the samples by RNA-Seq and used to perform between-group comparisons with non-parametric testing, multivariate logistic regression and classification analyses, as well as Monte-Carlo Cross-Validation (MCCV). The top miRNAs were examined for correlations with measures of adaptive behavior. Functional enrichment analysis of the highest confidence mRNA targets of the top differentially expressed miRNAs was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), as well as the Simons Foundation Autism Database (AutDB) of ASD candidate genes. Fourteen miRNAs were differentially expressed in ASD subjects compared to controls (p <0.05; FDR <0.15) and showed more than 95 % accuracy at distinguishing subject groups in the best-fit logistic regression model. MCCV revealed an average ROC-AUC value of 0.92 across 100 simulations, further supporting the robustness of the findings. Most of the 14 miRNAs showed significant correlations with Vineland neurodevelopmental scores. Functional enrichment analysis detected significant over-representation of target gene clusters related to transcriptional activation, neuronal development, and AutDB genes. Measurement of salivary miRNA in this pilot study of subjects with mild ASD demonstrated differential expression of 14 miRNAs that are expressed in the developing brain, impact mRNAs related to brain development, and correlate with neurodevelopmental measures of adaptive behavior. These miRNAs have high specificity and cross-validated utility as a potential screening tool for ASD.

Roles of microRNA in the immature immune system of neonates.

PubMed

Yu, Hong-Ren; Huang, Lien-Hung; Li, Sung-Chou

2018-06-13

Neonates have an immature immune system; therefore, their immune activities are different from the activities of adult immune systems. Such differences between neonates and adults are reflected by cell population constitutions, immune responses, cytokine production, and the expression of cellular/humoral molecules, which contribute to the specific neonatal microbial susceptibility and atopic properties. MicroRNAs (miRNAs) have been discovered to modulate many aspects of immune responses. Herein, we summarize the distinct manifestations of the neonatal immune system, including cellular and non-cellular components. We also review the current findings on the modulatory effects of miRNAs on the neonatal immune system. These findings suggest that miRNAs have the potential to be useful therapeutic targets for certain infection or inflammatory conditions by modulating the neonatal immune system. In the future, we need a more comprehensive understanding in regard to miRNAs and how they modulate specific immune cells in neonates. Copyright © 2018. Published by Elsevier B.V.

MicroRNA Expression Profiling of the Armed Forces Health Surveillance Branch Cohort for Identification of "Enviro-miRs" Associated With Deployment-Based Environmental Exposure.

PubMed

Dalgard, Clifton L; Polston, Keith F; Sukumar, Gauthaman; Mallon, Col Timothy M; Wilkerson, Matthew D; Pollard, Harvey B

2016-08-01

The aim of this study was to identify serum microRNA (miRNA) biomarkers that indicate deployment-associated exposures in service members at military installations with open burn pits. Another objective was to determine detection rates of miRNAs in Department of Defense Serum Repository (DoDSR) samples with a high-throughput methodology. Low-volume serum samples (n = 800) were profiled by miRNA-capture isolation, pre-amplification, and measurement by a quantitative PCR-based OpenArray platform. Normalized quantitative cycle values were used for differential expression analysis between groups. Assay specificity, dynamic range, reproducibility, and detection rates by OpenArray passed target desired specifications. Serum abundant miRNAs were consistently measured in study specimens. Four miRNAs were differentially expressed in the case deployment group subjects. miRNAs are suitable RNA species for biomarker discovery in the DoDSR serum specimens. Serum miRNAs are candidate biomarkers for deployment and environmental exposure in military service members.

A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures.

PubMed

Byron, Meg; Hall, Lisa L; Lawrence, Jeanne B

2013-01-01

Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

PubMed Central

Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

2014-01-01

SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

MicroRNA profiling of antler stem cells in potentiated and dormant states and their potential roles in antler regeneration.

PubMed

Ba, Hengxing; Wang, Datao; Li, Chunyi

2016-04-01

MicroRNAs (miRNAs) can effectively regulate gene expression at the post-transcriptional level and play a critical role in tissue growth, development and regeneration. Our previous studies showed that antler regeneration is a stem cell-based process and antler stem cells reside in the periosteum of a pedicle, the permanent bony protuberance, from which antler regeneration takes place. Antlers are the only mammalian organ that can fully regenerate and hence provide a unique opportunity to identify miRNAs that are involved in organ regeneration. In the present study, we used next generation sequencing technology sequenced miRNAs of the stem cells derived from either the potentiated or the dormant pedicle periosteum. A population of both conserved and 20 deer-specific miRNAs was identified. These conserved miRNAs were derived from 453 homologous hairpin precursors across 88 animal species, and were further grouped into 167 miRNA families. Among them, the miR-296 is embryonic stem cell-specific. The potentiation process resulted in the significant regulation (>±2 Fold, q value <0.05) of conserved miRNAs; 8 miRNA transcripts were down- and 6 up-regulated. Several GO biology processes and the Wnt, MAPK and TGF-beta signaling pathways were found to be up-regulated as part of antlerogenic stem cell potentiation process. This research has identified miRNAs that are associated either with the dormant or the potentiated antler stem cells and identified some target miRNAs for further research into their role played in mammalian organ regeneration.

The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1–3 synthesis

PubMed Central

Volkova, Eugenia; Gorchakov, Rodion; Frolov, Ilya

2008-01-01

Alphaviruses are regarded as attractive systems for expression of heterologous genes and development of recombinant vaccines. Venezuelan equine encephalitis virus (VEE)-based vectors are particularly promising because of their specificity to lymphoid tissues and strong resistance to interferon. To improve understanding of the VEE genome packaging and optimize application of this virus as a vector, we analyzed in more detail the mechanism of packaging of the VEE-specific RNAs. The presence of the RNAs in the VEE particles during serial passaging in tissue culture was found to depend not only on the presence of packaging signal(s), but also on the ability of these RNAs to express in cis nsP1, nsP2 and nsP3 in the form of a P123 precursor. Packaging of VEE genomes into infectious virions was also found to be more efficient compared to that of Sindbis virus, in spite of lower levels of RNA replication and structural protein production. PMID:16239019

Computational Analysis of Mouse piRNA Sequence and Biogenesis

PubMed Central

Betel, Doron; Sheridan, Robert; Marks, Debora S; Sander, Chris

2007-01-01

The recent discovery of a new class of 30-nucleotide long RNAs in mammalian testes, called PIWI-interacting RNA (piRNA), with similarities to microRNAs and repeat-associated small interfering RNAs (rasiRNAs), has raised puzzling questions regarding their biogenesis and function. We report a comparative analysis of currently available piRNA sequence data from the pachytene stage of mouse spermatogenesis that sheds light on their sequence diversity and mechanism of biogenesis. We conclude that (i) there are at least four times as many piRNAs in mouse testes than currently known; (ii) piRNAs, which originate from long precursor transcripts, are generated by quasi-random enzymatic processing that is guided by a weak sequence signature at the piRNA 5′ends resulting in a large number of distinct sequences; and (iii) many of the piRNA clusters contain inverted repeats segments capable of forming double-strand RNA fold-back segments that may initiate piRNA processing analogous to transposon silencing. PMID:17997596

Detection of piRNAs in whitespotted bamboo shark liver.

PubMed

Yang, Lingrong; Ge, Yinghua; Cheng, Dandan; Nie, Zuoming; Lv, Zhengbing

2016-09-15

Piwi-interacting RNAs (piRNAs) are 26 to 31-nt small non-coding RNAs that have been reported mostly in germ-line cells and cancer cells. However, the presence of piRNAs in the whitespotted bamboo shark liver has not yet been reported. In a previous study of microRNAs in shark liver, some piRNAs were detected from small RNAs sequenced by Solexa technology. A total of 4857 piRNAs were predicted and found in shark liver. We further selected 17 piRNAs with high and significantly differential expression between normal and regenerative liver tissues for subsequent verification by Northern blotting. Ten piRNAs were further identified, and six of these were matched to known piRNAs in piRNABank. The actual expression of six known and four novel piRNAs was validated by qRT-PCR. In addition, a total of 401 target genes of the 10 piRNAs were predicted by miRanda. Through GO and pathway function analyses, only five piRNAs could be annotated with eighteen GO annotations. The results indicated that the identified piRNAs are involved in many important biological responses, including immune inflammation, cell-specific differentiation and development, and angiogenesis. This manuscript provides the first identification of piRNAs in the liver of whitespotted bamboo shark using Solexa technology as well as further elucidation of the regulatory role of piRNAs in whitespotted bamboo shark liver. These findings may provide a useful resource and may facilitate the development of therapeutic strategies against liver damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and Characterization of Long Non-Coding RNAs Related to Mouse Embryonic Brain Development from Available Transcriptomic Data

PubMed Central

He, Hongjuan; Xiu, Youcheng; Guo, Jing; Liu, Hui; Liu, Qi; Zeng, Tiebo; Chen, Yan; Zhang, Yan; Wu, Qiong

2013-01-01

Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development. PMID:23967161

In situ amplification of intracellular microRNA with MNAzyme nanodevices for multiplexed imaging, logic operation, and controlled drug release.

PubMed

Zhang, Penghui; He, Zhimei; Wang, Chen; Chen, Jiangning; Zhao, Jingjing; Zhu, Xuena; Li, Chen-Zhong; Min, Qianhao; Zhu, Jun-Jie

2015-01-27

MicroRNAs (miRNAs), as key regulators in gene expression networks, have participated in many biological processes, including cancer initiation, progression, and metastasis, indicative of potential diagnostic biomarkers and therapeutic targets. To tackle the low abundance of miRNAs in a single cell, we have developed programmable nanodevices with MNAzymes to realize stringent recognition and in situ amplification of intracellular miRNAs for multiplexed detection and controlled drug release. As a proof of concept, miR-21 and miR-145, respectively up- and down-expressed in most tumor tissues, were selected as endogenous cancer indicators and therapy triggers to test the efficacy of the photothermal nanodevices. The sequence programmability and specificity of MNAzyme motifs enabled the fluorescent turn-on probes not only to sensitively profile the distributions of miR-21/miR-145 in cell lysates of HeLa, HL-60, and NIH 3T3 (9632/0, 14147/0, 2047/421 copies per cell, respectively) but also to visualize trace amounts of miRNAs in a single cell, allowing logic operation for graded cancer risk assessment and dynamic monitoring of therapy response by confocal microscopy and flow cytometry. Furthermore, through general molecular design, the MNAzyme motifs could serve as three-dimensional gatekeepers to lock the doxorubicin inside the nanocarriers. The drug nanocarriers were exclusively internalized into the target tumor cells via aptamer-guided recognition and reopened by the endogenous miRNAs, where the drug release rates could be spatial-temporally controlled by the modulation of miRNA expression. Integrated with miRNA profiling techniques, the designed nanodevices can provide general strategy for disease diagnosis, prognosis, and combination treatment with chemotherapy and gene therapy.

Genome wide predictions of miRNA regulation by transcription factors.

PubMed

Ruffalo, Matthew; Bar-Joseph, Ziv

2016-09-01

Reconstructing regulatory networks from expression and interaction data is a major goal of systems biology. While much work has focused on trying to experimentally and computationally determine the set of transcription-factors (TFs) and microRNAs (miRNAs) that regulate genes in these networks, relatively little work has focused on inferring the regulation of miRNAs by TFs. Such regulation can play an important role in several biological processes including development and disease. The main challenge for predicting such interactions is the very small positive training set currently available. Another challenge is the fact that a large fraction of miRNAs are encoded within genes making it hard to determine the specific way in which they are regulated. To enable genome wide predictions of TF-miRNA interactions, we extended semi-supervised machine-learning approaches to integrate a large set of different types of data including sequence, expression, ChIP-seq and epigenetic data. As we show, the methods we develop achieve good performance on both a labeled test set, and when analyzing general co-expression networks. We next analyze mRNA and miRNA cancer expression data, demonstrating the advantage of using the predicted set of interactions for identifying more coherent and relevant modules, genes, and miRNAs. The complete set of predictions is available on the supporting website and can be used by any method that combines miRNAs, genes, and TFs. Code and full set of predictions are available from the supporting website: http://cs.cmu.edu/~mruffalo/tf-mirna/ zivbj@cs.cmu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression profiling and bioinformatic analyses suggest new target genes and pathways for human hair follicle related microRNAs.

PubMed

Hochfeld, Lara M; Anhalt, Thomas; Reinbold, Céline S; Herrera-Rivero, Marisol; Fricker, Nadine; Nöthen, Markus M; Heilmann-Heimbach, Stefanie

2017-02-22

Human hair follicle (HF) cycling is characterised by the tight orchestration and regulation of signalling cascades. Research shows that micro(mi)RNAs are potent regulators of these pathways. However, knowledge of the expression of miRNAs and their target genes and pathways in the human HF is limited. The objective of this study was to improve understanding of the role of miRNAs and their regulatory interactions in the human HF. Expression levels of ten candidate miRNAs with reported functions in hair biology were assessed in HFs from 25 healthy male donors. MiRNA expression levels were correlated with mRNA-expression levels from the same samples. Identified target genes were tested for enrichment in biological pathways and accumulation in protein-protein interaction (PPI) networks. Expression in the human HF was confirmed for seven of the ten candidate miRNAs, and numerous target genes for miR-24, miR-31, and miR-106a were identified. While the latter include several genes with known functions in hair biology (e.g., ITGB1, SOX9), the majority have not been previously implicated (e.g., PHF1). Target genes were enriched in pathways of interest to hair biology, such as integrin and GnRH signalling, and the respective gene products showed accumulation in PPIs. Further investigation of miRNA expression in the human HF, and the identification of novel miRNA target genes and pathways via the systematic integration of miRNA and mRNA expression data, may facilitate the delineation of tissue-specific regulatory interactions, and improve our understanding of both normal hair growth and the pathobiology of hair loss disorders.

In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation.

PubMed

Hegde, Vikas; Hickerson, Robyn P; Nainamalai, Sitheswaran; Campbell, Paul A; Smith, Frances J D; McLean, W H Irwin; Pedrioli, Deena M Leslie

2014-12-28

Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

PubMed

Moyo, Lindani; Ramesh, Shunmugiah V; Kappagantu, Madhu; Mitter, Neena; Sathuvalli, Vidyasagar; Pappu, Hanu R

2017-07-17

Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

Circular RNAs: An emerging type of RNA in cancer.

PubMed

Hou, Li-Dan; Zhang, Jing

2017-03-01

Circular RNAs (circRNAs), a novel type of widespread and diverse endogenous non-coding RNAs (ncRNAs), which are different from the linear RNAs, form a covalently closed continuous loop without 5' or 3' polarities. The majority of circRNAs are abundant, conserved and stable across different species, and exhibit tissue/developmental-stage-specific characteristics. They are generated primarily through a type of alternative RNA splicing called "back-splicing," in which a downstream splice donor is joined to an upstream splice acceptor through splice skipping or direct splice. Recent studies have discovered circRNAs function as microRNA sponges, binding with RNA-associated proteins to form RNA-protein complexes and then regulating gene transcription and translation into polypeptides. Emerging evidence indicates that circRNAs play important roles in the regulation of the development and progression of multiple cancers by serving as potential diagnostic and predictive biomarkers involved in tumor growth and invasion and providing new strategies for cancer diagnosis and targeted therapy. In this review, we briefly delineate the diversity and characteristics of circRNAs and discuss the highlights of the biogenesis of circRNAs and their potential functions in tumor.

Role of Viral miRNAs and Epigenetic Modifications in Epstein-Barr Virus-Associated Gastric Carcinogenesis.

PubMed

Giudice, Aldo; D'Arena, Giovanni; Crispo, Anna; Tecce, Mario Felice; Nocerino, Flavia; Grimaldi, Maria; Rotondo, Emanuela; D'Ursi, Anna Maria; Scrima, Mario; Galdiero, Massimiliano; Ciliberto, Gennaro; Capunzo, Mario; Franci, Gianluigi; Barbieri, Antonio; Bimonte, Sabrina; Montella, Maurizio

2016-01-01

MicroRNAs are short (21-23 nucleotides), noncoding RNAs that typically silence posttranscriptional gene expression through interaction with target messenger RNAs. Currently, miRNAs have been identified in almost all studied multicellular eukaryotes in the plant and animal kingdoms. Additionally, recent studies reported that miRNAs can also be encoded by certain single-cell eukaryotes and by viruses. The vast majority of viral miRNAs are encoded by the herpesviruses family. These DNA viruses including Epstein-Barr virus encode their own miRNAs and/or manipulate the expression of cellular miRNAs to facilitate respective infection cycles. Modulation of the control pathways of miRNAs expression is often involved in the promotion of tumorigenesis through a specific cascade of transduction signals. Notably, latent infection with Epstein-Barr virus is considered liable of causing several types of malignancies, including the majority of gastric carcinoma cases detected worldwide. In this review, we describe the role of the Epstein-Barr virus in gastric carcinogenesis, summarizing the functions of the Epstein-Barr virus-encoded viral proteins and related epigenetic alterations as well as the roles of Epstein-Barr virus-encoded and virally modulated cellular miRNAs.

Expression profiling of snoRNAs in normal hematopoiesis and AML

PubMed Central

Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

2018-01-01

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

Characterization and Expression Patterns of microRNAs Involved in Rice Grain Filling

PubMed Central

Du, Yanxiu; Zhang, Jing; Li, Junzhou; Liu, Yanxia; Zhao, Yafan; Zhao, Quanzhi

2013-01-01

MicroRNAs (miRNAs) are upstream gene regulators of plant development and hormone homeostasis through their directed cleavage or translational repression of the target mRNAs, which may play crucial roles in rice grain filling and determining the final grain weight and yield. In this study, high-throughput sequencing was performed to survey the dynamic expressions of miRNAs and their corresponding target genes at five distinct developmental stages of grain filling. In total, 445 known miRNAs and 45 novel miRNAs were detected with most of them expressed in a developmental stage dependent manner, and the majority of known miRNAs, which increased gradually with rice grain filling, showed negatively related to the grain filling rate. Detailed expressional comparisons revealed a clear negative correlation between most miRNAs and their target genes. It was found that specific miRNA cohorts are expressed in a developmental stage dependent manner during grain filling and the known functions of these miRNAs are involved in plant hormone homeostasis and starch accumulation, indicating that the expression dynamics of these miRNAs might play key roles in regulating rice grain filling. PMID:23365650

Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination.

PubMed

Dong, Xiaomin; Chen, Kenian; Cuevas-Diaz Duran, Raquel; You, Yanan; Sloan, Steven A; Zhang, Ye; Zong, Shan; Cao, Qilin; Barres, Ben A; Wu, Jia Qian

2015-12-01

Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.

LncRNA and mRNA expression profiles of glioblastoma multiforme (GBM) reveal the potential roles of lncRNAs in GBM pathogenesis.

PubMed

Li, Qi; Jia, Hongmei; Li, Haowen; Dong, Chengya; Wang, Yajie; Zou, Zhongmei

2016-11-01

Glioblastoma multiforme (GBM) is the most common brain malignancy. Long non-coding RNAs (lncRNAs) are aberrantly expressed in many cancers and are involved in their cell proliferation, apoptosis, angiogenesis, and invasion. The functional roles of lncRNAs in GBM are less known. We analyzed a cohort of exon microarray datasets from The Cancer Genome Atlas. The differently expressed lncRNAs and mRNA were subjected to construct lncRNA-mRNA co-expression network. Probable functions for lncRNAs were predicted according to lncRNA-mRNA network and genomic adjacency by GO and pathway analysis. The expression of lncRNAs and mRNAs in GBM tissues versus normal brain tissues was examined by quantitative reverse transcription polymerase chain reaction. The 398 lncRNAs and 1995 mRNAs were identified as distinctively expressed in GBM. Probable functional roles for 98 lncRNAs were involved in 30 pathways and 32 gene functions related to tumorigenesis, development, and metastasis. The identified sets of key lncRNAs specific to GBM were subsequently verified by experiment in GBM tissues. Our reports predict the biological functions of a multitude of lncRNAs in GBM that could be potential diagnostic and prognostic biomarkers as well as therapeutic targets. Moreover, our research provides a road map for the identification and analysis of lncRNAs in tumors.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net; Tak, Hyosun, E-mail: chuberry@naver.com; Rho, Mina, E-mail: minarho@hanyang.ac.kr

2014-03-28

Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWImore » and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.« less

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

PubMed

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G; Rigoutsos, Isidore; Kirino, Yohei

2017-05-19

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Associations of circulating plasma microRNAs with age, body mass index and sex in a population-based study.

PubMed

Ameling, Sabine; Kacprowski, Tim; Chilukoti, Ravi Kumar; Malsch, Carolin; Liebscher, Volkmar; Suhre, Karsten; Pietzner, Maik; Friedrich, Nele; Homuth, Georg; Hammer, Elke; Völker, Uwe

2015-10-14

Non-cellular blood circulating microRNAs (plasma miRNAs) represent a promising source for the development of prognostic and diagnostic tools owing to their minimally invasive sampling, high stability, and simple quantification by standard techniques such as RT-qPCR. So far, the majority of association studies involving plasma miRNAs were disease-specific case-control analyses. In contrast, in the present study, plasma miRNAs were analysed in a sample of 372 individuals from a population-based cohort study, the Study of Health in Pomerania (SHIP). Quantification of miRNA levels was performed by RT-qPCR using the Exiqon Serum/Plasma Focus microRNA PCR Panel V3.M covering 179 different miRNAs. Of these, 155 were included in our analyses after quality-control. Associations between plasma miRNAs and the phenotypes age, body mass index (BMI), and sex were assessed via a two-step linear regression approach per miRNA. The first step regressed out the technical parameters and the second step determined the remaining associations between the respective plasma miRNA and the phenotypes of interest. After regressing out technical parameters and adjusting for the respective other two phenotypes, 7, 15, and 35 plasma miRNAs were significantly (q < 0.05) associated with age, BMI, and sex, respectively. Additional adjustment for the blood cell parameters identified 12 and 19 miRNAs to be significantly associated with age and BMI, respectively. Most of the BMI-associated miRNAs likely originate from liver. Sex-associated differences in miRNA levels were largely determined by differences in blood cell parameters. Thus, only 7 as compared to originally 35 sex-associated miRNAs displayed sex-specific differences after adjustment for blood cell parameters. These findings emphasize that circulating miRNAs are strongly impacted by age, BMI, and sex. Hence, these parameters should be considered as covariates in association studies based on plasma miRNA levels. The established experimental and computational workflow can now be used in future screening studies to determine associations of plasma miRNAs with defined disease phenotypes.

A highly sensitive SPRi biosensing strategy for simultaneous detection of multiplex miRNAs based on strand displacement amplification and AuNP signal enhancement.

PubMed

Wei, Xiaotong; Duan, Xiaolei; Zhou, Xiaoyan; Wu, Jiangling; Xu, Hongbing; Min, Xun; Ding, Shijia

2018-06-07

Herein, a dual channel surface plasmon resonance imaging (SPRi) biosensor has been developed for the simultaneous and highly sensitive detection of multiplex miRNAs based on strand displacement amplification (SDA) and DNA-functionalized AuNP signal enhancement. In the presence of target miRNAs (miR-21 or miR-192), the miRNAs could specifically hybridize with the corresponding hairpin probes (H) and initiate the SDA, resulting in massive triggers. Subsequently, the two parts of the released triggers could hybridize with capture probes (CP) and DNA-functionalized AuNPs, assembling DNA sandwiches with great mass on the chip surface. A significantly amplified SPR signal readout was achieved. This established biosensing method was capable of simultaneously detecting multiplex miRNAs with a limit of detection down to 0.15 pM for miR-21 and 0.22 pM for miR-192. This method exhibited good specificity and acceptable reproducibility. Moreover, the developed method was applied to the determination of target miRNAs in a complex matrix. Thus, this developed SPRi biosensing method may present a potential alternative tool for miRNA detection in biomedical research and clinical diagnosis.

Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina

PubMed Central

Damiani, Devid; Alexander, John J; O'Rourke, Jason R; McManus, Mike; Jadhav, Ashutosh P; Cepko, Constance L; Hauswirth, William W; Harfe, Brian D; Strettoi, Enrica

2009-01-01

MicroRNAs (miRNAs) are small, highly conserved molecules that have been shown to regulate the expression of genes by binding to specific target mRNAs. Dicer, an RNase III endonuclease, is essential for the production and function of mature miRNAs and removal of Dicer has been shown to disrupt many developmental processes. In this report, Dicer was removed specifically from the retina using a floxed Dicer conditional allele and the retinal Chx10Cre transgene. Retinal Dicer knockout mice displayed a reproducible inability to respond to light. In addition, morphological defects were observed with the formation of photoreceptor rosettes at P16 which progressed to more general cellular disorganization and widespread degeneration of retinal cell types as the animals aged. This was accompanied by concomitant decrease in both scotopic and photopic ERG responses. Interestingly, removing a single allele of Dicer resulted in ERG deficits throughout life but not to morphological abnormalities. Northern blot analysis of Dicer depleted retinas showed a decrease in several microRNAs. The observation that progressive retinal degeneration occurred upon removal of Dicer raises the possibility that miRNAs are involved in retinal neurodegenerative disorders. PMID:18463241

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

PubMed

Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2008-12-01

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

A novel class of small RNAs bind to MILI protein in mouse testes.

PubMed

Aravin, Alexei; Gaidatzis, Dimos; Pfeffer, Sébastien; Lagos-Quintana, Mariana; Landgraf, Pablo; Iovino, Nicola; Morris, Patricia; Brownstein, Michael J; Kuramochi-Miyagawa, Satomi; Nakano, Toru; Chien, Minchen; Russo, James J; Ju, Jingyue; Sheridan, Robert; Sander, Chris; Zavolan, Mihaela; Tuschl, Thomas

2006-07-13

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

Expression Profile of Long Noncoding RNAs in Peripheral Blood Mononuclear Cells from Multiple Sclerosis Patients.

PubMed

Zhang, Fang; Gao, Chao; Ma, Xiao-Feng; Peng, Xiao-Lin; Zhang, Rong-Xin; Kong, De-Xin; Simard, Alain R; Hao, Jun-Wei

2016-04-01

Long noncoding RNAs (lncRNAs) play a key role in regulating immunological functions. Their impact on the chronic inflammatory disease multiple sclerosis (MS), however, remains unknown. We investigated the expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) of patients with MS and attempt to explain their possible role in the process of MS. For this study, we recruited 26 patients with MS according to the revised McDonald criteria. Then, we randomly chose 6 patients for microarray analysis. Microarray assays identified outstanding differences in lncRNA expression, which were verified through real-time PCR. LncRNA functions were annotated for target genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and regulatory relationships between lncRNAs and target genes were analyzed using the "cis" and "trans" model. There were 2353 upregulated lncRNAs, 389 downregulated lncRNAs, 1037 upregulated mRNAs, and 279 downregulated mRNAs in patients with MS compared to healthy control subjects (fold change >2.0). Real-time PCR results of six aberrant lncRNAs were consistent with the microarray data. The coexpression network comprised 864 lncRNAs and 628 mRNAs. Among differentially expressed lncRNAs, 10 lncRNAs were predicted to have 10 cis-regulated target genes, and 33 lncRNAs might regulate their trans target genes. We identified a subset of dysregulated lncRNAs and mRNAs. The differentially expressed lncRNAs may be important in the process of MS. However, the specific molecular mechanisms and biological functions of these lncRNAs in the pathogenesis of MS need further study. © 2016 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

PubMed Central

Honda, Shozo; Kawamura, Takuya; Loher, Phillipe; Morichika, Keisuke; Rigoutsos, Isidore

2017-01-01

Abstract Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5′-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5′-tRNA halves as well, suggesting a previously uncharacterized link between 5′-tRNA halves and td-piRNAs. An increase in levels of the 5′-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5′-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to −1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs. PMID:28645172

MicroRNAs and non-coding RNAs in virus-infected cells

PubMed Central

Ouellet, Dominique L.; Provost, Patrick

2010-01-01

Within the past few years, microRNAs (miRNAs) and other non-coding RNAs (ncRNAs) have emerged as elements with critically high importance in post-transcriptional control of cellular and, more recently, viral processes. Endogenously produced by a component of the miRNA-guided RNA silencing machinery known as Dicer, miRNAs are known to control messenger RNA (mRNA) translation through recognition of specific binding sites usually located in their 3′ untranslated region. Recent evidences indicate that the host miRNA pathway may represent an adapted antiviral defense mechanism that can act either by direct miRNA-mediated modulation of viral gene expression or through recognition and inactivation of structured viral RNA species by the protein components of the RNA silencing machinery, such as Dicer. This latter process, however, is a double-edge sword, as it may yield viral miRNAs exerting gene regulatory properties on both host and viral mRNAs. Our knowledge of the interaction between viruses and host RNA silencing machineries, and how this influences the course of infection, is becoming increasingly complex. This review article aims to summarize our current knowledge about viral miRNAs/ncRNAs and their targets, as well as cellular miRNAs that are modulated by viruses upon infection. PMID:20217543

The Long Noncoding RNA Landscape of the Mouse Eye.

PubMed

Chen, Weiwei; Yang, Shuai; Zhou, Zhonglou; Zhao, Xiaoting; Zhong, Jiayun; Reinach, Peter S; Yan, Dongsheng

2017-12-01

Long noncoding RNAs (lncRNAs) are important regulators of diverse biological functions. However, an extensive in-depth analysis of their expression profile and function in mammalian eyes is still lacking. Here we describe comprehensive landscapes of stage-dependent and tissue-specific lncRNA expression in the mouse eye. Affymetrix transcriptome array profiled lncRNA signatures from six different ocular tissue subsets (i.e., cornea, lens, retina, RPE, choroid, and sclera) in newborn and 8-week-old mice. Quantitative RT-PCR analysis validated array findings. Cis analyses and Gene Ontology (GO) annotation of protein-coding genes adjacent to signature lncRNA loci clarified potential lncRNA roles in maintaining tissue identity and regulating eye maturation during the aforementioned phase. In newborn and 8-week-old mice, we identified 47,332 protein-coding and noncoding gene transcripts. LncRNAs comprise 19,313 of these transcripts annotated in public data banks. During this maturation phase of these six different tissue subsets, more than 1000 lncRNAs expression levels underwent ≥2-fold changes. qRT-PCR analysis confirmed part of the gene microarray analysis results. K-means clustering identified 910 lncRNAs in the P0 groups and 686 lncRNAs in the postnatal 8-week-old groups, suggesting distinct tissue-specific lncRNA clusters. GO analysis of protein-coding genes proximal to lncRNA signatures resolved close correlations with their tissue-specific functional maturation between P0 and 8 weeks of age in the 6 tissue subsets. Characterizating maturational changes in lncRNA expression patterns as well as tissue-specific lncRNA signatures in six ocular tissues suggest important contributions made by lncRNA to the control of developmental processes in the mouse eye.

miRNome analysis using real-time PCR.

PubMed

Pontrelli, Paola; Accetturo, Matteo; Gesualdo, Loreto

2014-01-01

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression in eukaryotic organisms, thus influencing physiological mechanisms such as development, cell proliferation, cell death, and cell differentiation. The importance of the gene regulatory system operated by miRNAs is emerging as a central topic in the setting of several diseases included infectious disease and cancer. The different techniques used for the study of the entire "miRNome" give the opportunity to go better inside these novel mechanisms of gene expression regulation. In the following method we describe a protocol based on quantitative real-time PCR (qRT-PCR) with SYBR(®) green technology, to specifically analyze the expression levels of only those miRNAs that target genes involved in CTLs biogenesis and functions. Through an in silico approach, we designed a custom microRNA qPCR panel focused on those miRNAs relevant in regulation of CTLs-specific pathways. The panel we created was customized by EXIQON, since this company proposed a method based on the use of LNA enhanced primers, which guarantee increased affinity and specificity for each microRNA. The advantage of this protocol with respect to a whole miRNome analysis consists in the possibility to evidence weaker signals that otherwise would be secreted and remove the noise itself generated by other miRNAs not directly involved in the regulation of CTLs-specific pathways. This panel can be applicable in the study of CTLs behavior in pathological conditions such as infectious disease and cancer or can be used to characterize changes in patients' immune responsiveness after therapeutic intervention in order to understand the molecular mechanisms underlying these effects.

EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research.

PubMed

Kim, Dae-Kyum; Lee, Jaewook; Simpson, Richard J; Lötvall, Jan; Gho, Yong Song

2015-04-01

For cell-to-cell communication, all living cells including archaea, bacteria, and eukaryotes secrete nano-sized membrane vesicles into the extracellular space. These extracellular vesicles harbor specific subsets of proteins, mRNAs, miRNAs, lipids, and metabolites that represent their cellular status. These vesicle-specific cargos are considered as novel diagnostic biomarkers as well as therapeutic targets. With the advancement in high-throughput technologies on multiomics studies and improvements in bioinformatics approaches, a huge number of vesicular proteins, mRNAs, miRNAs, lipids, and metabolites have been identified, and our understanding of these complex extracellular organelles has considerably increased during these past years. In this review, we highlight EVpedia (http://evpedia.info), a community web portal for systematic analyses of prokaryotic and eukaryotic extracellular vesicles research. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcripts with in silico predicted RNA structure are enriched everywhere in the mouse brain

PubMed Central

2012-01-01

Background Post-transcriptional control of gene expression is mostly conducted by specific elements in untranslated regions (UTRs) of mRNAs, in collaboration with specific binding proteins and RNAs. In several well characterized cases, these RNA elements are known to form stable secondary structures. RNA secondary structures also may have major functional implications for long noncoding RNAs (lncRNAs). Recent transcriptional data has indicated the importance of lncRNAs in brain development and function. However, no methodical efforts to investigate this have been undertaken. Here, we aim to systematically analyze the potential for RNA structure in brain-expressed transcripts. Results By comprehensive spatial expression analysis of the adult mouse in situ hybridization data of the Allen Mouse Brain Atlas, we show that transcripts (coding as well as non-coding) associated with in silico predicted structured probes are highly and significantly enriched in almost all analyzed brain regions. Functional implications of these RNA structures and their role in the brain are discussed in detail along with specific examples. We observe that mRNAs with a structure prediction in their UTRs are enriched for binding, transport and localization gene ontology categories. In addition, after manual examination we observe agreement between RNA binding protein interaction sites near the 3’ UTR structures and correlated expression patterns. Conclusions Our results show a potential use for RNA structures in expressed coding as well as noncoding transcripts in the adult mouse brain, and describe the role of structured RNAs in the context of intracellular signaling pathways and regulatory networks. Based on this data we hypothesize that RNA structure is widely involved in transcriptional and translational regulatory mechanisms in the brain and ultimately plays a role in brain function. PMID:22651826

The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

PubMed

Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A Aziz

2013-01-01

Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

The CCR4-NOT Complex Mediates Deadenylation and Degradation of Stem Cell mRNAs and Promotes Planarian Stem Cell Differentiation

PubMed Central

Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A. Aziz

2013-01-01

Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology. PMID:24367277

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

PubMed

Max, Klaas E A; Bertram, Karl; Akat, Kemal Marc; Bogardus, Kimberly A; Li, Jenny; Morozov, Pavel; Ben-Dov, Iddo Z; Li, Xin; Weiss, Zachary R; Azizian, Azadeh; Sopeyin, Anuoluwapo; Diacovo, Thomas G; Adamidi, Catherine; Williams, Zev; Tuschl, Thomas

2018-06-05

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies. Copyright © 2018 the Author(s). Published by PNAS.

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

PubMed Central

Langlois, Ryan A; Shapiro, Jillian S; Pham, Alissa M; tenOever, Benjamin R

2012-01-01

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery. PMID:22086233

MicroRNAs in large herpesvirus DNA genomes: recent advances.

PubMed

Sorel, Océane; Dewals, Benjamin G

2016-08-01

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that regulate gene expression. They alter mRNA translation through base-pair complementarity, leading to regulation of genes during both physiological and pathological processes. Viruses have evolved mechanisms to take advantage of the host cells to multiply and/or persist over the lifetime of the host. Herpesviridae are a large family of double-stranded DNA viruses that are associated with a number of important diseases, including lymphoproliferative diseases. Herpesviruses establish lifelong latent infections through modulation of the interface between the virus and its host. A number of reports have identified miRNAs in a very large number of human and animal herpesviruses suggesting that these short non-coding transcripts could play essential roles in herpesvirus biology. This review will specifically focus on the recent advances on the functions of herpesvirus miRNAs in infection and pathogenesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen

Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression,more » and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection.« less

A novel program to design siRNAs simultaneously effective to highly variable virus genomes.

PubMed

Lee, Hui Sun; Ahn, Jeonghyun; Jun, Eun Jung; Yang, Sanghwa; Joo, Chul Hyun; Kim, Yoo Kyum; Lee, Heuiran

2009-07-10

A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.

Long non-coding RNAs as regulators of the endocrine system

PubMed Central

Knoll, Marko; Lodish, Harvey F.; Sun, Lei

2015-01-01

Long non-coding RNAs (lncRNAs) are a large and diverse group of RNAs that are often lineage-specific and that regulate multiple biological functions. Many are nuclear and are essential parts of ribonucleoprotein complexes that modify chromatin segments and establish active or repressive chromatin states; others are cytosolic and regulate the stability of mRNA or act as microRNA sponges. This Review summarizes the current knowledge of lncRNAs as regulators of the endocrine system, with a focus on the identification and mode of action of several endocrine-important lncRNAs. We highlight lncRNAs that have a role in the development and function of pancreatic β cells, white and brown adipose tissue, and other endocrine organs, and discuss the involvement of these molecules in endocrine dysfunction (for example, diabetes mellitus). We also address the associations of lncRNAs with nuclear receptors involved in major hormonal signalling pathways, such as estrogen and androgen receptors, and the relevance of these associations in certain endocrine cancers. PMID:25560704

Long non-coding RNAs as regulators of the endocrine system.

PubMed

Knoll, Marko; Lodish, Harvey F; Sun, Lei

2015-03-01

Long non-coding RNAs (lncRNAs) are a large and diverse group of RNAs that are often lineage-specific and that regulate multiple biological functions. Many are nuclear and are essential parts of ribonucleoprotein complexes that modify chromatin segments and establish active or repressive chromatin states; others are cytosolic and regulate the stability of mRNA or act as microRNA sponges. This Review summarizes the current knowledge of lncRNAs as regulators of the endocrine system, with a focus on the identification and mode of action of several endocrine-important lncRNAs. We highlight lncRNAs that have a role in the development and function of pancreatic β cells, white and brown adipose tissue, and other endocrine organs, and discuss the involvement of these molecules in endocrine dysfunction (for example, diabetes mellitus). We also address the associations of lncRNAs with nuclear receptors involved in major hormonal signalling pathways, such as estrogen and androgen receptors, and the relevance of these associations in certain endocrine cancers.

The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism.

PubMed

Makeyev, E V; Bamford, D H

2000-11-15

Bacteriophage φ6 has a three-segmented double-stranded (ds) RNA genome, which resides inside a polymerase complex particle throughout the entire life cycle of the virus. The polymerase subunit P2, a minor constituent of the polymerase complex, has previously been reported to replicate both φ6-specific and heterologous single-stranded (ss) RNAs, giving rise to dsRNA products. In this study, we show that the enzyme is also able to use dsRNA templates to perform semi-conservative RNA transcription in vitro without the assistance of other proteins. The polymerase synthesizes predominantly plus-sense copies of φ6 dsRNA, medium and small segments being more efficient templates than the large one. This distribution of the test-tube reaction products faithfully mimics viral transcription in vivo. Experiments with chimeric ssRNAs and dsRNAs show that short terminal nucleotide sequences can account for the difference in efficiency of RNA synthesis. Taken together, these results suggest a model explaining important aspects of viral RNA metabolism regulation in terms of enzymatic properties of the polymerase subunit.

Epigenetic Regulation of miRNAs and Breast Cancer Stem Cells

PubMed Central

Duru, Nadire; Gernapudi, Ramkishore; Eades, Gabriel; Eckert, Richard; Zhou, Qun

2015-01-01

MicroRNAs have emerged as important targets of chemopreventive strategies in breast cancer. We have found that miRNAs are dysregulated at an early stage in breast cancer, in non-malignant Ductal Carcinoma In Situ. Many dietary chemoprevention agents can act by epigenetically activating miRNA-signaling pathways involved in tumor cell proliferation and invasive progression. In addition, many miRNAs activated via chemopreventive strategies target cancer stem cell signaling and prevent tumor progression or relapse. Specifically, we have found that miRNAs regulate DCIS stem cells, which may play important roles in breast cancer progression to invasive disease. We have shown that chemopreventive agents can directly inhibit DCIS stem cells and block tumor formation in vivo, via activation of tumor suppressor miRNAs. PMID:26052481

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

PubMed

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

Noncoding RNAs in Neurodegenerative Diseases

PubMed Central

Rege, Shraddha D.; Geetha, Thangiah; Pondugula, Satyanarayana R.; Zizza, Claire A.; Wernette, Catherine M.

2013-01-01

Noncoding RNAs are widely known for their various essential roles in the development of central nervous system. It involves neurogenesis, neural stem cells generation, maintenance and maturation, neurotransmission, neural network plasticity, formation of synapses, and even brain aging and DNA damage responses. In this review, we will discuss the biogenesis of microRNA, various functions of noncoding RNA's specifically microRNAs (miRNAs) that act as the chief regulators of gene expression, and focus in particular on misregulation of miRNAs which leads to several neurodegenerative diseases as well as its therapeutic outcome. Recent evidences has shown that miRNAs expression levels are changed in patients with neurodegenerative diseases; hence, miRNA can be used as a potential diagnostic biomarker and serve as an effective therapeutic tool in overcoming various neurodegenerative disease processes. PMID:23738143

RNA Interference: Biology, Mechanism, and Applications

PubMed Central

Agrawal, Neema; Dasaradhi, P. V. N.; Mohmmed, Asif; Malhotra, Pawan; Bhatnagar, Raj K.; Mukherjee, Sunil K.

2003-01-01

Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. PMID:14665679

Small-Interfering RNA–Eluting Surfaces as a Novel Concept for Intravascular Local Gene Silencing

PubMed Central

Nolte, Andrea; Walker, Tobias; Schneider, Martina; Kray, Oya; Avci-Adali, Meltem; Ziemer, Gerhard; Wendel, Hans Peter

2011-01-01

New drug-eluting stent (DES) methods have recently been demonstrated to improve outcomes of intravascular interventions. A novel technique is the design of gene-silencing stents that elute specific small-interfering RNAs (siRNAs) for better vascular wall regeneration. Although siRNAs used to alter gene expression have surpassed expectations in in vitro experiments, the functional and local delivery of siRNAs is still the major obstacle for the in vivo application of RNA interference. In this preliminary in vitro study we investigated a surface-immobilized siRNA delivery technique that would be readily adaptable for local intravascular applications in vivo. The transfection potency of gelatin coatings consisting of a specific siRNA complexed with polyethylenimine (PEI) was examined in primary human endothelial cells by flow cytometry and quantitative real-time polymerase chain reaction. Several media conditions, such as the presence or absence of serum during cultivation, were investigated. Furthermore, different siRNA and PEI amounts, as well as nitrogen/phosphate ratios, were tested for their transfection efficiency. Gelatin coatings consisting of PEI and siRNA against an exemplary endothelial adhesion molecule receptor achieved a significant knockdown of around 70%. The transfection efficiency of the coatings was not influenced by the presence of serum. The results of this preliminary study support the expectation that this novel coating may be favorable for local in vivo gene silencing (for example, when immobilized on stents or balloons for percutanous transluminal coronary angioplasty). However, further animal experiments are needed to confirm the translation into clinical practice. This intriguing technology leads the way to more sophisticated and individualized coatings for the post-DES era, toward silencing of genes involved in the pathway of intimal hyperplasia. PMID:21792480

Biogenesis pathways of piRNAs loaded onto AGO3 in the Drosophila testis.

PubMed

Nagao, Akihiro; Mituyama, Toutai; Huang, Haidong; Chen, Dahua; Siomi, Mikiko C; Siomi, Haruhiko

2010-12-01

PIWI-interacting RNAs (piRNAs) silence transposable elements in animal germ cells. In Drosophila ovaries, piRNAs are produced by two distinct pathways: the "ping-pong" amplification cycle that operates in germ cells and a ping-pong-independent pathway termed the primary pathway that mainly operates in somatic cells. AGO3, one of three PIWI proteins in flies, is involved in the ping-pong cycle in ovaries. We characterized AGO3-associated piRNAs in fly testes and found that like in ovaries, AGO3 functions in the ping-pong cycle with Aubergine (Aub) for piRNA production from transposon transcripts. In contrast, most AGO3-associated piRNAs corresponding to Suppressor of Stellate [Su(Ste)] genes are antisense-oriented and bound to Aub. In addition, the vast majority of AGO3-bound piRNAs derived from the AT-chX locus on chromosome X are antisense-oriented and are also found among Aub-associated piRNAs. The presence of very few sense Su(Ste) and AT-chX piRNAs suggests that biogenesis of both Su(Ste) and AT-chX piRNAs by a ping-pong mechanism only is highly unlikely. Nevertheless, the mutual interdependence of AGO3 and Aub for the accumulation of these piRNAs shows that their production relies on both AGO3 and Aub. Analysis of piRNA pathway mutants revealed that although the requirements for piRNA factors for Su(Ste)- and AT-chX-piRNA levels mostly overlap and resemble those for the ping-pong mechanism in the ovaries, Armitage (armi) is not required for the accumulation of AT-chX-1 piRNA. These findings suggest that the impacts of armi mutants on the operation of the piRNA pathway are variable in germ cells of fly testes.

A Macro View of MicroRNAs: The Discovery of MicroRNAs and Their Role in Hematopoiesis and Hematologic Disease

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2017-01-01

MicroRNAs (miRNAs) are a class of endogenously encoded ~22 nucleotide, noncoding, single-stranded RNAs that contribute to development, body planning, stem cell differentiation, and tissue identity through posttranscriptional regulation and degradation of transcripts. Given their importance, it is predictable that dysregulation of miRNAs, which target a wide variety of transcripts, can result in malignant transformation. In this review, we explore the discovery of miRNAs, their mechanism of action, and the tools that aid in their discovery and study. Strikingly, many of the studies that have expanded our understanding of the contributions of miRNAs to normal physiology and in the development of diseases have come from studies in the hematopoietic system and hematologic malignancies, with some of the earliest identified functions for mammalian miRNAs coming from observations made in leukemias. So, with a special focus on the hematologic system, we will discuss how miRNAs contribute to differentiation of stem cells and how dysregulation of miRNAs contributes to the development of malignancy, by providing examples of specific miRNAs that function as oncogenes or tumor suppressors, as well as of defects in miRNA processing. Finally, we will discuss the promise of miRNA-based therapeutics and challenges for the future study of disease-causing miRNAs. PMID:28838543

Detection and Analysis of Circular RNAs by RT-PCR.

PubMed

Panda, Amaresh C; Gorospe, Myriam

2018-03-20

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

Ultrasensitive and Multiple Disease-Related MicroRNA Detection Based on Tetrahedral DNA Nanostructures and Duplex-Specific Nuclease-Assisted Signal Amplification.

PubMed

Xu, Fang; Dong, Haifeng; Cao, Yu; Lu, Huiting; Meng, Xiangdan; Dai, Wenhao; Zhang, Xueji; Al-Ghanim, Khalid Abdullah; Mahboob, Shahid

2016-12-14

A highly sensitive and multiple microRNA (miRNA) detection method by combining three-dimensional (3D) DNA tetrahedron-structured probes (TSPs) to increase the probe reactivity and accessibility with duplex-specific nuclease (DSN) for signal amplification for sensitive miRNA detection was proposed. Briefly, 3D DNA TSPs labeled with different fluorescent dyes for specific target miRNA recognition were modified on a gold nanoparticle (GNP) surface to increase the reactivity and accessibility. Upon hybridization with a specific target, the TSPs immobilized on the GNP surface hybridized with the corresponding target miRNA to form DNA-RNA heteroduplexes, and the DSN can recognize the formed DNA-RNA heteroduplexes to hydrolyze the DNA in the heteroduplexes to produce a specific fluorescent signal corresponding to a specific miRNA, while the released target miRNA strands can initiate another cycle, resulting in a significant signal amplification for sensitive miRNA detection. Different targets can produce different fluorescent signals, leading to the development of a sensitive detection for multiple miRNAs in a homogeneous solution. Under optimized conditions, the proposed assay can simultaneously detect three different miRNAs in a homogeneous solution with a logarithmic linear range spanning 5 magnitudes (10 -12 -10 -16 ) and achieving a limit of detection down to attomolar concentrations. Meanwhile, the proposed miRNA assay exhibited the capability of discriminating single bases (three bases mismatched miRNAs) and showed good eligibility in the analysis of miRNAs extracted from cell lysates and miRNAs in cell incubation media, which indicates its potential use in biomedical research and clinical analysis.