The human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) are a versatile Gram-positive cocci that havw shown complex modes of regulation of its different virulence factors. Discoveries of a few small non-coding RNAs (sRNAs) in S. pyogenes and their influence on the expression of virulence factors revealed an important role of sRNAs on S. pyogenes virulence. The genome-wide analysis of bacterial genomes for the discovery of sRNAs through computational methods has become an effective way to discover new sRNAs. In this study we provided a computational scheme where three different algorithms (RNAz, eQRNA, and sRNAPredict) were combined to increase the probabilities of predicting putative sRNAs within S. pyogenes' intergenic regions (IGR). A total of 46 candidates were chosen based on our criteria, and through Northern blot we analyzed each candidate. We obtained hybridization signals from twelve newly discovered sRNAs in S. pyogenes. Subsequently, we analyzed their sequence and their location within the IGR to find a putative -10 promoter region and possible Rho-independent terminator site, and their possible targets through computational methods. We further expanded our analysis of the new sRNAs by using Real-Time RT-PCR to determine the expression of sRNAs during different phases of growth. Our results showed that our computational scheme and experimental method was effective in predicting sRNAs previously undiscovered in S. pyogenes, and that more sRNAs are yet to be discovered and characterized, helping to further understand the regulation of virulence factors in S. pyogenes

PART1, TITLE: A p53-inducible long non-coding RNA PICART1 mediating cancer cell proliferation and migration. Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs are characterized thus far. A novel lncRNA transcript with 2.53 kb in length was identified by a transcriptome sequencing analysis, named p53-inducible cancer-associated RNA transcript 1 (PICART1). This PICART1 is upregulated by p53 through a p53-binding site at -1808 to -1783bp. In breast and colorectal cancer cells and tissues, PICART1 expression was decreased. Ectopic expression of the PICART1 suppressed growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated the cell growth and migration. In these cells, the expression of PICART1 lowered down the levels of p-AKT (Thr308 & Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, but p21cip1/Waf1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade. PART2, TITLE: The novel long non-coding RNA PANCR is a tumor suppressor gene in breast cancer. Long non-coding RNAs (lncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new lncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR lncRNA consists of 1.5kb in length. Our data showed that PANCR was downregulated in breast cancer tissues and cell lines. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31 (62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 cells, ectopic expression of PANCR suppressed cell proliferation in culture, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation.

Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria.

Le cholangiocarcinome intra hépatique (CCI) est une tumeur hépatique primitive développée aux dépens des canaux biliaires. Son pronostic est mauvais avec les traitements actuels qui augmentent peu la survie des patients. Sa cancérogénèse est complexe impliquant de nombreuses voies de signalisation dont la voie TGFβ. L’hypothèse du projet est l’implication des ARN longs non-codants (ARNlnc) comme médiateurs de la voie TGFβ dans le développement du CCI. Les objectifs de notre travail étaient d’identifier des ARNlnc régulés par le TGFβ et potentiels biomarqueurs diagnostiques ou pronostiques. Nous avons identifié une signature transcriptomique spécifique du TGFβ après stimulation de lignées cellulaires de CCI. Parmi les nouveaux gènes cibles, plusieurs ARNlnc ont été identifiés dont CASC15 renommé TLINC pour TGFβ-induced long intergenic non-coding RNA. TLINC aurait un rôle dans le remodelage du microenvironnement impliqué dans la cancérogénèse du CCI notamment par la régulation de l’IL8. Ce rôle pourrait s’exercer par l’interaction avec d’autres ARNlnc déjà identifiés dans le CCI e.g. NEAT1. TLINC est surexprimé dans les tumeurs humaines de CCI et pourrait constituer un biomarqueur diagnostique. Des isoformes circulaires de TLINC mises en évidence dans les tumeurs pourraient être détectables dans le sérum et constituer des biomarqueurs non invasifs. L’analyse transcriptomique d’une cohorte de patients divisée en 2 sous-groupes de pronostic différent a identifié une signature d’ARNlnc prédictive de la survie. L’ARNlnc ANRIL, déjà connu dans d’autres cancers, est un des ARNlnc qui pourrait constituer un biomarqueur pronostique. / Intrahepatic cholangiocarcinoma (ICC) is a primary liver tumor developed from bile ducts. ICC prognosis is poor with current treatments that slightly increase patient survival. ICC carcinogenesis is complex and involves multiple signaling pathways including TGFβ pathway. Our hypothesis relies on the involvement of long non-coding RNA (lncRNA) as mediators of TGFβ pathway in the development of ICC. The aim of the study was to identify and to characterize TGFβ regulated lncRNA as ICC potential diagnostic or prognostic biomarkers. We identified a specific transcriptomic signature after stimulation of ICC cell lines with TGFβ. Among the novel TGFβ target genes, several lncRNAs were identified including CASC15 renamed TLINC standing for TGFβ-induced long intergenic non-coding RNA. TLINC may play a role in the remodeling of an inflammatory microenvironment involved in ICC carcinogenesis, including the regulation of IL8. This role could be exerted by the interaction with other lncRNAs already identified in the ICC e.g. NEAT1. TLINC is overexpressed in human ICC tumors and may represent a relevant diagnostic biomarker. Circular isoforms of TLINC found in tumors may be detectable in serum and be noninvasive biomarkers. Transcriptomic analysis of tumors from a cohort of patients divided into 2 prognostic groups identified a lncRNAs signature predictive for survival. LncRNA ANRIL, already known to be upregulated in other cancers, is one of the lncRNAs that could be a prognostic biomarker in ICC.

Polycomb Proteins (PcG) are chromatin proteins that control the maintenance of “transcriptional memory” and cell identity by fixing the repressed state of developmentally regulated genes. This function has been linked to interaction with RNA moieties, in particular long non-coding RNAs (lncRNAs). However, specificity of PcG-RNA interactions has been controversial (Beltran et al., 2016; Chen Davidovich, Leon Zheng, Karen J. Goodrich, & Thomas R. Cech, 2013). In this study we took advantage of recent work published from our lab reporting about a novel and reversible mechanism regulating genome wide Ezh1-PRC2 activation in mouse skeletal muscle cells in response to atrophic stress (Bodega et al., 2017). Using this physiological, in vivo tool we could identify a functional dynamic crosstalk between Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and PRC2-Ezh1 complex. By combining immuno-fluorescence, biochemistry, epigenomics, ChIRP, DNA and RNA immunoprecipitation we identified a novel pathway in which Malat1 plays a role in compartmentalization, assembly and activity of PRC2 in chromatin, allowing epigenetic plastic response to atrophic stress and recovery. We conclude that Malat1 is an essential partner for PRC2-Ezh1 adaptive function in skeletal muscle cells.

Chromatin associated RNAs play a key role in mediating epigenetic mechanism. To improve our comprehension of how chromatin-associated RNAs influence gene expression, it is crucial to identify the genomic locus that RNA interacts with on a genome-wide scale. Emerging technologies were developed to capture binding sites of lncRNAs globally. Such techniques rely on the concept of Proximity ligation via a biotinylated adapter to ligate DNA and RNA on each end, such as MARGI, GRID-seq, ChAR-seq and the most recent technology, devised in our lab RNA And DNA Complexes Ligated & Sequenced (RADICL-seq). RADICL-seq has several advantages over the other methods. However, while this method produced a fairly global picture of the chromatin associated “RNA-ome”, the specific association with specific regulatory factors is missing, in addition all emerging technologies lack the sensitivity to capture low-expressed RNAs. Thus, we set out a strategy to address this issue by enriching global RNA-chromatin interactions mediated by a specific factor which will be achieved by combining RADICL with Chromatin Immunoprecipitation (ChIP) method. Since our lab interest is investigating the role of lncRNAs in muscle differentiation, RADICL-ChIP has been performed using the C2C12 mouse skeletal muscle. In particular, the role of identification of RNA moieties interacting with PRC2 PcG proteins in stress response and their genome wide distribution in chromatin.

Regulation and function of protein-coding genes are increasingly well-understood, but no comparable evidence exists for non-coding RNA (ncRNA) genes, which appear to be more numerous than protein-coding genes. We developed a novel machine-learning model to distinguish promoters of long ncRNA (lncRNA) genes from those of protein-coding genes. This represents the first attempt to make this distinction based on properties of the associated gene promoters. From our analyses, several transcription factors (TFs), which are known to be regulated by lncRNAs, also emerged as potential global regulators of lncRNAs, suggesting that lncRNAs and TFs may participate in bidirectional feedback regulatory network. Our results also raise the possibility that, due to the historical dependence on protein-coding gene in defining the chromatin states of active promoters, an adjustment of these chromatin signature profiles to incorporate lncRNAs is warranted in the future. Secondly, we developed a novel method to infer functions for lncRNA and microRNA (miRNA) transcripts based on their transcriptional regulatory networks in 119 tissues and 177 primary cells of human. This method for the first time combines information of cell/tissueVspecific expression of a transcript and the TFs and transcription coVfactors (TcoFs) that control activation of that transcript. Transcripts were annotated using statistically enriched GO terms, pathways and diseases across cells/tissues and associated knowledgebase (FARNA) is developed. FARNA, having the most comprehensive function annotation of considered ncRNAs across the widest spectrum of cells/tissues, has a potential to contribute to our understanding of ncRNA roles and their regulatory mechanisms in human. Thirdly, we developed a novel machine-learning model to identify LD motif (a protein interaction motif) of paxillin, a ncRNA target that is involved in cell motility and cancer metastasis. Our recognition model identified new proteins not previously known to harbor LD motifs and we experimentally confirmed some of our predicted motifs. This novel discovery will expand our knowledge of cancer metastasis and will facilitate therapeutic targeting linking specific ncRNAs via paxillin proteins to diseases. Finally, through bioinformatics approaches, we identified lncRNAs as markers that distinguish classical from alternative activation of macrophage. This result may have good use in the diagnosis of infectious diseases.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Bc. Magdaléna Krhutová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: The changes in expression of long non-coding RNAs in hepatocellular carcinoma Hepatocellular carcinoma (HCC) is one of the highly prevalent cancers globally. A number of new cases of HCC and deaths rises every year. Molecular mechanisms of HCC are being intensively studied, yet they are not still fully understood. In addition to genetic alterations, epigenetics also plays an important role in HCC pathogenesis. Long non- coding RNAs (lncRNAs) are RNA molecules that are not capable of coding proteins, and their length is 200 nucleotides or more. Various studies have already revealed lncRNAs involved in tumorigenesis through binding to DNA, RNA and proteins. New studies also demonstrate significant changes in the expression of biotransformation enzymes in HCC, and interactions with microRNAs (miRNAs) and lncRNAs. This diploma thesis deals with the issue of long non-coding RNAs in relation to HCC. It summarizes the epidemiological situation, risk factors, and current possibilities of diagnosis and therapy of this disease. It also summarizes recently described genetic and epigenetic mechanisms contributing...

Non-coding RNA (ncRNA) therapeutics are based on short oligonucleotides, both naturally occurring and artificial, which target RNA in a site-specific way to modulate gene expression. As of today, 12 synthetically produced ncRNA-based drugs are available on the market in the US and Europe, and there is a possibility of more to be approved in the near future. This project is ordered by Cytiva, a global life science company, with the aim to present an overview of the current ncRNA therapeutics field. The aim is to give Cytiva a clear indication of what type of products for oligonucleotide synthesis are requested by their clients in the pharmaceutical industry. ncRNAs were examined extensively for their potential as therapeutic agents during our literature study. Based on the number of approved drugs, clinical trials, and our overall impression of future potential, we selected the following four ncRNAs; Antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and small nuclear RNA (snRNA). Among these, the most promising ncRNAs for therapeutic use are siRNA and ASO which usually are 20-30 nucleotides long. The most common modifications to improve drug-like properties are modifications to the backbone, sugar modifications at position 2, and methylation of nucleobases at position 5 of the oligonucleotide. In addition, ncRNA-based drugs on the market today are delivered either through non-viral mechanisms or without a delivery system. The conclusion that can be drawn from our report is the importance of being able to synthesize chemically modified ASOs and siRNA on a large scale to meet the future demand of the pharmaceutical industry.

We comprehensively examined the rare variants in the IPO11-HTR1A region to explore their roles in neuropsychiatric disorders. Five hundred seventy-three to 1,181 rare SNPs in subjects of European descent and 1,234-2,529 SNPs in subjects of African descent (0