Search

International audience; Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.

Background:Evidence has demonstrated that non-coding RNAs (ncRNAs) could be delivered efficiently to recipient cells using exosomes as a carrier. Additionally, long ncRNA nuclear enriched abundant transcript 1 (NEAT1) is emerging as a vital regulatory molecule in the progression of rheumatoid arthritis (RA). The aim of this study was to identify the NEAT1/miR-144-3p/Rho-associated protein kinase 2 (ROCK2) functional network regulating the WNT signaling pathway in RA.Methods:In vivo, a collagen-induced arthritis (CIA) model was established to analyze the effects of blood exosomes on the incidence, clinical score, and bone degradation of RA. In vitro, the CD4+T cells were characterized by flow cytometry and the cell activities were analyzed in the presence of exosome treatment alone or in combination with altered expression of NEAT1, miR-144-3p or Rho-associated protein kinase 2 (ROCK2). The expression of NEAT1, miR-144-3p, ROCK2, and corresponding proteins in the WNT signaling pathway was detected by RT-qPCR and western blot techniques. The binding profile of NEAT1 to miR-144-3p was evaluated via a combination approach of luciferase activity assay, RNA immunoprecipitation, and RNA pull-down experiments.Results:Blood exosomes extracted from RA patients increased the incidence of RA and bone destruction significantly. Overexpression of NEAT1 or ROCK2 promoted immune cell (CD4+T cells) proliferation, Th17 cell differentiation, and cell migration in response to stimulus, whereas knockout of the NEAT1 gene induced the expression of miR-144-3p in CD4+T cells. ROCK2 exogenous expression inhibited the expression of miR-144-3p, inducing activation of the WNT signaling pathway.Conclusion:A novel regulatory pathway NEAT1/miR-144-3p/ROCK2/WNT in RA was investigated as a potential target for RA therapy.

It has been demonstrated that RNA G-quadruplexes (G4) are structural motifs present in transcriptomes and play important regulatory roles in several post-transcriptional mechanisms. However, the full picture of RNA G4 locations and the extent of their implication remain elusive. Solely computational prediction analysis of the whole transcriptome may reveal all potential G4, since experimental identifications are always limited to specific conditions or specific cell lines. The present study reports the first in-depth computational prediction of potential G4 region across the complete human transcriptome. Although using a relatively stringent approach based on three prediction scores that accounts for the composition of G4 sequences, the composition of their neighboring sequences, and the various forms of G4, over 1.1 million of potential G4 (pG4) were predicted. The abundance of G4 was computationally confirmed in both 5′ and 3′UTR as well as splicing junction of mRNA, appreciate for the first time in the long ncRNA, while almost absent of most of the small ncRNA families. The present results constitute an important step toward a full understanding of the roles of G4 in post-transcriptional mechanisms.

Yellow lupine (Lupinus luteus L.) belongs to a legume family that benefits from symbiosis with nitrogen-fixing bacteria. Its seeds are rich in protein, which makes it a valuable food source for animals and humans. Yellow lupine is also the model plant for basic research on nodulation or abscission of organs. Nevertheless, the knowledge about the molecular regulatory mechanisms of its generative development is still incomplete. The RNA-Seq technique is becoming more prominent in high-throughput identification and expression profiling of both coding and non-coding RNA sequences. However, the huge amount of data generated with this method may discourage other scientific groups from making full use of them. To overcome this inconvenience, we have created a database containing analysis-ready information about non-coding and coding L. luteus RNA sequences (LuluDB). LuluDB was created on the basis of RNA-Seq analysis of small RNA, transcriptome, and degradome libraries obtained from yellow lupine cv. Taper flowers, pod walls, and seeds in various stages of development, flower pedicels, and pods undergoing abscission or maintained on the plant. It contains sequences of miRNAs and phased siRNAs identified in L. luteus, information about their expression in individual samples, and their target sequences. LuluDB also contains identified lncRNAs and protein-coding RNA sequences with their organ expression and annotations to widely used databases like GO, KEGG, NCBI, Rfam, Pfam, etc. The database also provides sequence homology search by BLAST using, e.g., an unknown sequence as a query. To present the full capabilities offered by our database, we performed a case study concerning transcripts annotated as DCL 1–4 (DICER LIKE 1–4) homologs involved in small non-coding RNA biogenesis and identified miRNAs that most likely regulate DCL1 and DCL2 expression in yellow lupine. LuluDB is available at http://luluseqdb.umk.pl/basic/web/index.php.

In this study, Wang et al. demonstrate that lncRNA-encoded polypeptide ASRPS is down-regulated in TNBC. ASRPS regulates angiogenesis and may serve as a novel prognostic marker and therapeutic target for TNBC., Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BC) with the most aggressive phenotype and poor overall survival. Using bioinformatics tools, we identified LINC00908 encoding a 60–aa polypeptide and differentially expressed in TNBC tissues. We named this endogenously expressed polypeptide ASRPS (a small regulatory peptide of STAT3). ASRPS expression was down-regulated in TNBCs and associated with poor overall survival. We showed that LINC00908 was directly regulated by ERα, which was responsible for the differential down-regulation of LINC00908 in TNBCs. ASRPS directly bound to STAT3 through the coiled coil domain (CCD) and down-regulated STAT3 phosphorylation, which led to reduced expression of VEGF. In human endothelial cells, a mouse xenograft breast cancer model, and a mouse spontaneous BC model, ASRPS expression reduced angiogenesis. In a mouse xenograft breast cancer model, down-regulation of ASRPS promoted tumor growth, and ASRPS acted as an antitumor peptide. We presented strong evidence that LINC00908-encoded polypeptide ASRPS represented a TNBC-specific target for treatment., Graphical Abstract

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington’s disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.

Spermatogonial stem cells (SSCs), a unique population of male germ cells with self-renewal ability, are the foundation for maintenance of spermatogenesis throughout the life of the male. Although many regulatory molecules essential for SSC self-renewal have been identified, the fundamental mechanism underlying how SSCs acquire and maintain their self-renewal activity remains largely to be elucidated. In recent years, many types of noncoding RNAs (ncRNAs) have been suggested to regulate the SSC self-renewal through multiple ways, indicating ncRNAs play crucial roles in SSC self-renewal. In this paper, we mainly focus on four types of ncRNAs including microRNA, long ncRNA, piwi-interacting RNA, as well as circular RNAs, and reviewed their potential roles in SSC self-renewal that discovered recently to help us gain a better understanding of molecular mechanisms by which ncRNAs perform their function in regulating SSC self-renewal.

Noncoding RNAs (ncRNAs), primarily microRNAs and long ncRNAs, play important roles in lung cancer. However, the role of long ncRNA (lncRNA)-X-inactive specific transcript (XIST) in non-small-cell lung cancer (NSCLC) is unclear. The purpose of this study was to explore the biologic function and potential mechanism of XIST in NSCLC progression.XIST, miR-137, and Notch-1 expression were detected by quantitative real-time PCR (qRT-PCR). Levels of proliferation- and epithelial-mesenchymal transition (EMT)-related proteins were assessed by Western blot. The correlations between XIST and miR-137, as well as miR-137 and Notch-1, were evaluated by bioinformatic analysis and luciferase reporter assays.We confirmed that XIST is aberrantly upregulated in NSCLC tissues and cell lines. XIST depletion inhibited cell proliferation and TGF-β1-induced EMT in A549 and H1299 cells. Spearman's correlation analysis showed an inverse correlation between miR-137 and XIST in NSCLC tissues, and miR-137 levels were found to be aberrantly reduced in A549 and H1299 cells. Furthermore, XIST could act as an endogenous sponge by directly binding to miR-137, negatively regulating its expression. miR-137 overexpression inhibited proliferation and TGF-β1-induced EMT in A549 and H1299 cells, whereas XIST could reverse the inhibitory effect of miR-137 on proliferation and TGF-β1-induced EMT. In addition, Notch-1 was identified as a direct target gene of miR-137, with the XIST-miR-137 axis regulating activation of the Notch-1 pathway.We identified a branch of the XIST/miR-137/Notch-1 pathway that regulates proliferation and TGF-β1-induced EMT in NSCLC, which could be involved in NSCLC progression.

Most of the human genome consists of DNA genes that are translated into RNAs but not into proteins. These RNA molecules are named noncoding RNAs (ncRNA). While in the past it was thought that ncRNAs would be redundant without relevant functions, it is now well established that ncRNAs identify a class of regulatory molecules that finely tune cell homeostasis and are deregulated in disease states, including hepatocellular carcinoma (HCC). Of note, the number of ncRNAs within a cell increases progressively, with the complexity of the species indicating their essential role in the maintenance of regulatory networks that affect the intricacy of the organism. ncRNAs have been demonstrated to mediate HCC development and progression by affecting intrinsic cancer cell signaling and crosstalk between malignant cells and the microenvironment. Moreover, ncRNAs hold promise as clinical biomarkers, but further evidence is warranted before their translation and integration within clinical practice.

Regulation of microRNA (miRNA) expression has been extensively studied with respect to colorectal cancer (CRC), since CRC is one of the leading causes of cancer mortality worldwide. Transcriptional control of miRNAs creating clusters can be, to some extent, estimated from cluster position on a chromosome. Levels of miRNAs are also controlled by miRNAs “sponging” by long non-coding RNAs (ncRNAs). Both types of miRNA regulation strongly influence their function. We focused on clusters of miRNAs found to be down-regulated in CRC, containing miR-1, let-7, miR-15, miR-16, miR-99, miR-100, miR-125, miR-133, miR-143, miR-145, miR-192, miR-194, miR-195, miR-206, miR-215, miR-302, miR-367 and miR-497 and analysed their genome position, regulation and functions. Only evidence provided with the use of CRC in vivo and/or in vitro models was taken into consideration. Comprehensive research revealed that down-regulated miRNA clusters in CRC are mostly located in a gene intron and, in a majority of cases, miRNA clusters possess cluster-specific transcriptional regulation. For all selected clusters, regulation mediated by long ncRNA was experimentally demonstrated in CRC, at least in one cluster member. Oncostatic functions were predominantly linked with the reviewed miRNAs, and their high expression was usually associated with better survival. These findings implicate the potential of down-regulated clusters in CRC to become promising multi-targets for therapeutic manipulation.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that are not translated into proteins. They have recently gained widespread attention due to the finding that tens of thousands of lncRNAs reside in the human genome, and due to an increasing number of lncRNAs that are found to be associated with disease. Some lncRNAs, including disease-associated ones, play different roles in regulating the cell cycle. Mathematical models of the cell cycle have been useful in better understanding this biological system, such as how it could be robust to some perturbations and how the cell cycle checkpoints could act as a switch. Here, we discuss mathematical modeling techniques for studying lncRNA regulation of the mammalian cell cycle. We present examples on how modeling via network analysis and differential equations can provide novel predictions toward understanding cell cycle regulation in response to perturbations such as DNA damage.

Background Long non-coding RNAs (lncRNAs) represent a novel class of non-coding RNAs having a crucial role in many biological processes. The identification of long non-coding homologs among different species is essential to investigate such roles in model organisms as homologous genes tend to retain similar molecular and biological functions. Alignment–based metrics are able to effectively capture the conservation of transcribed coding sequences and then the homology of protein coding genes. However, unlike protein coding genes the poor sequence conservation of long non-coding genes makes the identification of their homologs a challenging task. Results In this study we compare alignment–based and alignment–free string similarity metrics and look at promoter regions as a possible source of conserved information. We show that promoter regions encode relevant information for the conservation of long non-coding genes across species and that such information is better captured by alignment–free metrics. We perform a genome wide test of this hypothesis in human, mouse, and zebrafish. Conclusions The obtained results persuaded us to postulate the new hypothesis that, unlike protein coding genes, long non-coding genes tend to preserve their regulatory machinery rather than their transcribed sequence. All datasets, scripts, and the prediction tools adopted in this study are available at https://github.com/bioinformatics-sannio/lncrna-homologs. Electronic supplementary material The online version of this article (10.1186/s12859-018-2441-6) contains supplementary material, which is available to authorized users.

Frontiers in pharmacology 9, 805 (2018). doi:10.3389/fphar.2018.00805, Published by Frontiers Media, Lausanne

Gastric cancer (GC) is among the most frequent malignant diseases. Despite advances in treatment, the clinical outcome of patients with GC remains poor. The establishment of novel biomarkers is urgently required for early detection, treatment evaluation and prognostic assessment. Non-coding RNAs (ncRNAs) are a key topic of intensive research due to their potential applications in the field of oncology. The long ncRNA H19 has been frequently reported as overexpressed in many cancers including GC. In the present study, the diagnostic and prognostic value of circulating H19 in GC was assessed. Higher levels of circulating H19 were identified in GC patients (n=40) compared with a control group consisting of endoscopy-verified GC-free individuals (n=42; median levels relative to GAPDH, 58.4 vs. 29.9; P=0.027). Patients with smaller tumor sizes (

Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs—a long ncRNA, a circular RNA, and two microRNAs—using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7–targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network. Three different classes of noncoding RNA converge in a regulatory network whereby a long noncoding RNA represses a microRNA via target-directed microRNA degradation, which in turn enables the accumulation of a circular RNA in the mouse brain., NIH (Grants GM061835, GM118135 and T32CA009216)

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA., G.A.C. is The Alan M. Gewirtz Leukemia & Lymphoma Society Scholar. Work in G.A.C.’s laboratory is supported in part by the NIH/NCI grants 1UH2TR00943-01 and 1 R01 CA182905-01, the UT MD Anderson Cancer Center SPORE in Melanoma grant from NCI (P50 CA093459), Aim at Melanoma Foundation and the Miriam and Jim Mulva research funds, the Brain SPORE (2P50CA127001), the Center for Radiation Oncology Research Project, the Center for Cancer Epigenetics Pilot project, a 2014 Knowledge GAP MDACC grant, a CLL Moonshot pilot project, the UT MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment, a SINF grant in colon cancer, the Laura and John Arnold Foundation, the RGK Foundation, and the Estate of C.G. Johnson, Jr. I.B.-N. was financed by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR. S.M.G.D., A.L.B.A., and D.A. are supported by the São Paulo Research Foundation FAPESP under grants 2014/15968-3, 2014/20673-2, and 2014/17820-3, respectively. W.L. was partly supported by grants from The University of Texas MD Anderson Cancer Center Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Research. J.A.B. was supported by the Cancer Center Support Grant (P30 CA016672), and the HP imaging program of the Small Animal Facility (SAIF) was supported by the Cancer Prevention and Research Institutes of Texas grant RP-101243P5. H.L. was supported by NIH/NCI grant R01CA175486, a grant (RP140462) from the Cancer Prevention and Research Institute of Texas, and the R. Lee Clark Fellow Award from The Jeanne F. Shelby Scholarship Fund. I.B.-N. was financed by a Fulbright fellowship and by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR.

Background Long noncoding RNAs (lncRNA) are key regulators of gene transcription and many show tissue-specific expression. We previously defined a novel inflammatory and metabolic ileal gene signature in treatment-naive pediatric Crohn disease (CD). We now extend our analyses to include potential regulatory lncRNA. Methods Using RNAseq, we systematically profiled lncRNAs and protein-coding gene expression in 177 ileal biopsies. Co-expression analysis was used to identify functions and tissue-specific expression. RNA in situ hybridization was used to validate expression. Real-time polymerase chain reaction was used to test lncRNA regulation by IL-1β in Caco-2 enterocytes. Results We characterize widespread dysregulation of 459 lncRNAs in the ileum of CD patients. Using only the lncRNA in discovery and independent validation cohorts showed patient classification as accurate as the protein-coding genes, linking lncRNA to CD pathogenesis. Co-expression and functional annotation enrichment analyses across several tissues and cell types 1showed that the upregulated LINC01272 is associated with a myeloid pro-inflammatory signature, whereas the downregulated HNF4A-AS1 exhibits association with an epithelial metabolic signature. We confirmed tissue-specific expression in biopsies using in situ hybridization, and validated regulation of prioritized lncRNA upon IL-1β exposure in differentiated Caco-2 cells. Finally, we identified significant correlations between LINC01272 and HNF4A-AS1 expression and more severe mucosal injury. Conclusions We systematically define differentially expressed lncRNA in the ileum of newly diagnosed pediatric CD. We show lncRNA utility to correctly classify disease or healthy states and demonstrate their regulation in response to an inflammatory signal. These lncRNAs, after mechanistic exploration, may serve as potential new tissue-specific targets for RNA-based interventions.

Colorectal cancer (CRC) is one of the greatest threats to public health. Recent advances in whole-genome transcriptome analyses have enabled the identification of numerous members of a novel class of non-coding (nc)RNA, long ncRNA (lncRNA), which is broadly defined as RNA molecules that are >200 nt in length and lacking an open reading frame. In the present review, all lncRNAs associated with CRC are briefly summarized, with a particular focus on their potential roles as clinical biomarkers. CRC-associated lncRNAs involved in the underlying mechanisms of CRC progression are also initially included. This should benefit the development of novel markers and effective therapeutic targets for patients with CRC.

Long non-coding RNAs (ncRNAs) control almost every level of the gene expression program adding an unexpected layer of complexity in the regulation of gene expression. They have been shown to control fundamental biological processes such as X chromosome inactivation, imprinting, proliferation, development and differentiation. Furthermore, they are involved in the development of a wide variety of human disorders such as cancer and neurodegenerative disorders. Recent research revealed that some long ncRNAs are expressed from a subset of enhancers and are required for mediating their function. However, the extent to which long ncRNAs are required for enhancer function is still unknown. Additionally, although enhancers have been studied for decades, there is still no consensus on how to predict tissue-specific enhancers. In this thesis we employed a recently developed methodology- PreSTIGE to predict tissue specific enhancers and their targets based on the tissue specific presence of H3K4me1 marks and tissue specific gene expression. We find that 28 % (2,695) of all ENCODE annotated long ncRNAs overlap tissue- specific enhancers predicted by PreSTIGE. The expression of enhancer overlapping long ncRNAs is significantly higher in a tissue in which an overlapping enhancer is predicted to be active suggesting that some enhancers might require long ncRNAs for their activity. This dependency for long ncRNA expression is not observed at enhancers predicted by a different methodology. Additionally, we find that enhancers expressing long ncRNAs have a lower H3K4me1/H3K4me3 ratio suggesting that they might have a specific epigenetic profile. In summary, we verify the tissue-specific predictive power of PreSTIGE and demonstrate that almost one third of long ncRNAs are expressed from tissue-specific enhancers suggesting that the interplay between long ncRNAs and enhancers is important for regulation of tissue-specific gene expression. Although we are able to detect thousands of long ncRNAs due to the technological progress identifying functional long ncRNAs and functional characterization of these low abundant transcripts is still challenging. By using functional data for differential expression of long ncRNAs in differentiating keratinocytes and RNA polymerase II association, we identified PARROT, a functional long ncRNA expressed at a relatively high level in HeLa cells. Genome wide transcriptome and proteome analysis upon depletion of PARROT revealed that PARROT acts as an upstream regulator of c-Myc affecting cellular proliferation, migration and translation. Furthermore, we find that PARROT is down-regulated in senescence and up-regulated in some cancers further suggesting that PARROT has an important role in the regulation of cellular proliferation., Lange nicht-kodierende RNAs (ncRNAs) kontrollieren nahezu jede Ebene des Genexpressionsprogramms und fügen diesem eine unerwartete Komplexität hinzu. NcRNAs kontrollieren fundamentale biologische Prozesse wie z.B. X-Chromosom- Inaktivierung, Imprinting, Zellteilung, Entwicklung und Differenzierung. Zusätzlich sind sie involviert in der Entstehung einer Vielzahl von menschlichen Krankheiten wie Krebs oder neurodegenerative Erkrankungen. Jüngste Forschungsergebnisse haben gezeigt, dass einige lange ncRNAs von einer Teilmenge der Enhancerelemente im menschlichen Genom exprimiert werden und notwendig sind um die Enhancerfunktion zu vermitteln. Dennoch sind das Ausmaß zu welchem lange ncRNAs für die Enhancerfunktion benötigt werden noch nicht bekannt. Zusätzlich besteht bisher kein Konsens darüber wie Gewebs-spezifische Enhancer vorher gesagt werden sollten obwohl Enhancer bereits seit Jahrzehnten erforscht werden. In dieser Doktorarbeit verwenden wir eine kürzlich entwickelte Methode – PreSTIGE – um Gewebs-spezifische Enhancer sowie ihre Zielgene vorherzusagen. Diese Methode basiert auf dem Gewebs-spezifischen Vorhandensein von Histonmethylierungen, im speziellen H3K4me1, sowie der Gewebs-spezifischen Genexpression. Dabei finden wir, dass 28% (2.695) aller ENCODE annotierten langen ncRNAs mit Gewebs-spezifischen Enhancern überlappen, die als solche durch PreSTIGE vorhergesagt werden. Die Expression von solchen langen ncRNAs, die mit Enhancern überlappen, ist signifikant höher im jeweiligen Gewebe in dem der entsprechende Enhancer vorhergesagt wird aktiv zu sein. Dies deutet darauf hin, dass einige Enhancer die Expression von langen ncRNAs zur Ausführung ihrer Enhancerfunktion benötigen. Diese Abhängigkeit zur gleichzeitigen Expression von langen ncRNAs wird nicht beobachtet an Enhancern, die mittels einer anderen Methode vorausgesagt werden. Zusätzlich finden wir, dass Enhancer, die eine lange ncRNA exprimieren, ein geringeres Verhältnis von H3K4me1/H3K4me3 Histonmodifikationen aufweisen, was darauf hindeutet, dass sie ein spezifisches epigenetisches Profil aufweisen könnten. Zusammenfassend weisen wir die Gewebs- spezifische Vorhersagekraft von Enhancern durch PreSTIGE nach und zeigen außerdem, dass fast ein Drittel aller annotierten long ncRNAs von Gewebs- spezifischen Enhancern transkribiert werden. Dies deutet darauf hin, dass das Zusammenspiel von langen ncRNAs und Enhancern wichtig ist für die Regulation von Gewebs-spezifischer Genexpression. Trotz des technologischen Fortschritts tausende lange ncRNAs zu detektieren, ist die Identifizierung funktionaler langer ncRNAs sowie die funktionelle Charakterisierung dieser gering exprimierten Transkripte anspruchsvoll. Mittels Verwendung funktioneller Daten für die differentielle Expression von langen ncRNAs in differenzierenden Keratinocyten und deren Assoziation mit RNA Polymerase II, identifizieren wir PARROT, eine funktionale lange ncRNA, die zu relativ hohen Leveln in HeLa Zellen exprimiert ist. Genomweite Transkriptom- und Proteomanalyse im Anschluss an den knock-down von PARROT zeigte, dass PARROT als ein vorgeschalteter Regulator von c-Myc fungiert und aufgrund dessen Zellteilung, Migration und Translation in HeLa Zellen beeinflusst. Weiterhin beobachten wir, dass die Expression von PARROT herunterreguliert ist während zellulärer Seneszenz und heraufreguliert ist in einigen Krebsarten. Damit könnte PARROT eine wichtige Rolle in der Regulation der zellulären Proliferation zugeschrieben werden.

The past decade has seen dramatic changes in our understanding of the scale and complexity of eukaryotic transcriptome owing to the discovery of diverse types of short and long non-protein-coding RNAs (ncRNAs). While short ncRNA-mediated gene regulation has been extensively studied and the mechanisms well understood, the function of long ncRNAs remains largely unexplored, especially in plants. Nevertheless, functional insights generated in recent studies with mammalian systems have indicated that long ncRNAs are key regulators of a variety of biological processes. They have been shown to act as transcriptional regulators and competing endogenous RNAs (ceRNAs), to serve as molecular cargos for protein re-localization and as modular scaffolds to recruit the assembly of multiple protein complexes for chromatin modifications. Some of these functions have been found to be conserved in plants. Here, we review our current understanding of long ncRNA functions in plants and discuss the challenges in functional characterization of plant long ncRNAs.

Many protein-coding gene (PCG) promoters in the human genome initiate transcription in two directions, thereby expressing an mRNA and an upstream non-coding RNA (ncRNA). Diverse species of these promoter-associated ncRNAs are abundantly detected in genome-wide transcriptome studies but the functions of these non-coding transcripts remain mostly elusive. In this thesis, a set of 1,107 long ncRNA/PCG pairs that are expressed from bidirectional promoters is defined. These bidirectional promoters exhibit a high degree of sequence conservation and mediate linked expression of paired genes. This is determined by expression quantification and reporter assays of selected candidates. Expression of these long ncRNA/PCG pairs is detected to frequently occur from promoters of cancer-related proteins. One of the bidirectional promoters mediates simultaneous expression of the tumor suppressor gene RB1 and ncRNA- RB1 as detected by assaying the effects of retinoblastoma-associated point mutations in a bidirectional reporter assay. The linked expression of both genes is further shown by mutation of core promoter elements residing in both promoter directions. Changes of single or few base-pairs, is found to affect transcription initiation in both promoter directions equally. To determine the functionality of paired genes and their involvement in common biological pathways, ncRNA-RB1 and RB1 mRNA were individually depleted in a cell culture system. This revealed that both genes are not regulating each other’s expression and that ncRNA-RB1 conveys regulatory effects that are different but also to a certain degree overlapping to the RB1 controlled transcriptional program. NcRNA-RB1 positively regulates the expression of calreticulin (CALR), an endoplasmic reticulum-sessile chaperone that can translocate to the surface of tumor cells after chemotherapy, thereby serving as an ’eat-me-signal’ to phagocytes. Knock-down of the nuclear-retained ncRNA-RB1 in tumor cells reduces the expression of the CALR gene on chromatin, impairs translocation of the CALR protein to the cell surface upon treatment with anthracylines, and consequently inhibits uptake of the cells by macrophages. In conclusion, co- transcription of ncRNA-RB1 from the bidirectional RB1 promoter provides a positive link between the regulation of two the tumor suppressors RB1 and CALR. Loss of expression of either gene product of the ncRNA-RB1/RB1 pair entails the abolition of additional tumor-inhibitory mechanisms., Viele Promotoren Protein-kodierender Gene im menschlichen Genom initiieren die Transkription in zwei Richtungen und exprimieren dabei eine Boten-RNA (mRNA) sowie eine nicht-kodierende RNA (ncRNA), welche upstream des Promoters liegt. Verschiedene Varianten solcher Promoter-assoziierten, ncRNAs wurden kürzlich in Genom-weiten Transkriptionsstudien detektiert. Dennoch sind ihre Funktionen bisher weitgehend ungeklärt. In dieser Doktorarbeit, wird ein Set bestehend aus 1107 Paaren langer ncRNAs (>200 bp) und Protein-kodierender Gene definiert, die von bidirektionellen Promotoren exprimiert werden. Entsprechende Promotoren weisen ein hohes Maß an Sequenzkonservierung auf und initiieren gleichzeitig die Expression von Genpaaren. Durch Quantifizierung der Expression und Verwendung von Reporter Assays für ausgewählte Kandidatengenpaare wurde dieses Verhalten nachgewiesen. Viele dieser bidirektionellen Promotoren exprimieren Gene, die im Zusammenhang zur Entstehung von Tumoren stehen. Einer dieser Promotoren vermittelt die gleichzeitige Expression des Tumorsuppressors RB1 und der ncRNA-RB1. Dieses wird mittels eines Reporter-Assays gezeigt, welcher die Auswirkungen von Retinoblastoma-assoziierten Punktmutationen auf die Bidirektionionalität des Promotors nachvollzieht. Weiterhin wird die gekoppelte Expression der Genpaare durch Mutation von Core-Promoterelementen gezeigt, welche sich in beide Richtungen des bidirektionellen ncRNA-RB1/RB1 Promoters befinden. Dabei beeinflusste die artifizielle Veränderung einzelner oder einiger weniger Basenpaare die Transkriptionsinitiation in beide Promoterrichtungen. Um die Funktionalität beider Gene eines Genpaares sowie ihre Beteiligung in gemeinsamen biologischen Stoffwechselwegen aufzuklären, wurden die ncRNA-RB1 und die RB1 mRNA einzeln inaktiviert. Dieser Versuch zeigte, dass beide Gene nicht gegenseitig ihre Expression beeinflussen und die ncRNA-RB1 regulatorische Effekte besitzt, die unterschiedlich von, andererseits aber auch überlappend mit der transkriptionellen Regulation durch RB1 sind. Unabhängig von RB1 beeinflusst die ncRNA-RB1 die Expression von Calreticulin (CALR), eines Chaperons des endoplasmatischen Retikulums, positiv. Nach Behandlung mit spezifischen Chemotherapeutika kann CALR zur Zelloberfläche von Tumorzellen translozieren und dort als Fress-Signal für phagozytierende Zellen dienen. Der Knock-down der nukleären ncRNA-RB1 in Tumorzellen reduziert die Transkription des CALR-Gens und verhindert nachfolgend die Translokation des CALR-Proteins zur Zelloberfläche als Auswirkung der Behandlung mit Anthracyclinen. Die Konsequenz daraus ist eine verhinderte Aufnahme der ncRNA- RB1 knock-down Zellen durch Makrophagen. Als Ergebnis stellt die gleichzeitige Transkription von ncRNA-RB1 und RB1 von einem gemeinsamen bidirektionalen Promoter eine Verknüpfung zwischen der Regulation der zwei Tumorsuppressoren RB1 und CALR her. Der Verlust der Expression jedes Gens des Paares ncRNA- RB1/RB1 führt zur Beeinträchtigung Tumor unterdrückender Mechanismen in der Zelle.

In this book, a brief history and survey of the state of the art in the field of data integration, modeling and pattern identification with respect to pathway mining and presented a number of potential and challenging directions in which it could be extended. The prospects of the application of bioinformatics to pathway analysis have attracted increasing attentions in the past two decades due to the importance of pathway in understanding complex regulatory networks of biology systems. In recent years, these approaches and techniques have been showing their maturity and consolidation. A lot of specialized or general-purpose algorithms and tools have been proposed and used to realistic pathway data analysis, in many cases generating interesting and valuable biology knowledge to biologist that can be further applied to enhance and prompt the relevant study of life science, such as disease diagnosis and drug design. However, it is necessary for us to recognize that some emerging challenges, such as new and complex systems biology and meta-genome and new types of regulatory factors, such as long ncRNA, give rise to new requirements to existing pathway data analysis. Any efforts to discover and construct pathwaysmust taken them into account. These give rise some a series of important issues that need to be investigated for improving the correctness and efficiency of pathway mining.

A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes., Singapore-MIT Alliance for Research and Technology, National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES002109)

The majority of the mammalian genome is transcribed into long non-coding (lnc) RNAs, transcripts longer than 200 nucleotides and devoid of protein-coding potential. Recent genome-wide mapping effor...