Many classes of non-coding RNAs (ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum) can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.

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A plethora of new functions of non-coding RNAs have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this \Modern RNA World' and its components. In this contribution we attempt to provide at least a cursory overview of the diversity of non-coding RNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates, studies of the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of microRNAs in metazoans, which suggests an explosive increase in the microRNA repertoire in vertebrates. The analysis of the transcription of non-coding RNAs (ncRNAs) suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA.

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.

Wound healing is a complex physiological process. Effective wound healing enables the skin barrier function to be restored once the skin is injured. However, due to the complex nature of wounds, the mechanisms underlying tissue repair are still poorly understood. This has hindered the development of treatment for chronic wound, which is posing threat to both human health system and economy. Long non-coding RNAs (lncRNAs) have been identified as important gene expression regulators and to play functional roles in many biological processes.  The aim of this study was to unravel the gene regulatory network in human skin wound healing, in particular, to identify lncRNAs that may play a functional role in skin repair. Here we performed RNA sequencing to profile gene expression in fibroblasts and keratinocytes isolated from matched skin and day-7 acute wounds of five healthy donors. We predicted a total of 1974 and 3444 mRNA–lncRNA correlated pairs in wound fibroblasts and wound keratinocytes, respectively. By integrating the results from gene ontology enrichment and weighted co-expression network analysis, we shortlisted lncRNAs that may play a functional role in human skin wound healing.

Ancestral environmental conditions can instruct offspring development, although the mechanism(s) underlying such transgenerational epigenetic inheritance is unclear. In murine models focused on paternal dietary effects, we and others have identified tRNA fragments (tRFs) in mature sperm as potential carriers of epigenetic information. In our search for molecular targets of specific tRFs, we observed that altering the level of 5’-tRF Glycine-GCC (tRF-GG) in mouse embryonic stem cells (mESCs) and preimplantation embryos modulates the expression of the endogenous retrovirus MERV-L and genes regulated by MERV-L. Intriguingly, transient derepression of MERV-L is associated with totipotency of two-cell stage embryos and a subset of two-cell-like mESCs. Here, I reveal the mechanistic basis for tRF-GG regulation of MERV-L. I show that tRF-GG supports the production of numerous small nuclear RNAs associated with the Cajal body, in mouse and human embryonic stem cells. In particular, tRF-GG modulates the levels of U7 snRNA to ensure an adequate supply of histone proteins. This in turn safeguards heterochromatin-mediated transcriptional repression of MERV-L elements. Importantly, tRF-GG effects on histone mRNA levels, activity of a histone 3’UTR reporter, and expression of MERV-L associated transcripts can all be suppressed by appropriate manipulation of U7 RNA levels. I also show that hnRNPF and H bind directly to tRF-GG, and display a stark overlap of in vivo functions to tRF-GG. Together, this data uncovers a conserved mechanism for a 5’ tRNA fragment in the fine-tuning of a regulatory cascade to modulate global chromatin organization during pre-implantation development.

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Indiana University-Purdue University Indianapolis (IUPUI) / Non-coding RNAs across the genome have been associated with various biological processes, ranging from regulation of splicing to remodeling of chromatin. Amongst the repertoire of non-coding sequences lies a critical species of RNAs called long non-coding RNAs (lncRNAs). LncRNAs significantly contribute to a large spectrum of human phenotypes, including cancers, Heart failure, Diabetes, and Alzheimer’s disease. This dissertation emphasizes the need to characterize the functional role of lncRNAs to improve our understanding of human diseases. This work consolidates a resource from multiple computational genomics and natural language processing-based approaches to advance our ability to functionally annotate hundreds of lncRNAs and their interactions, providing a one-stop lncRNA functional annotation and dynamic interaction network and multi-facet omics data visualization platform. RNA interactions are vital in various cellular processes, from transcription to RNA processing. These interactions dictate the functional scope of the RNA. However, the multifaceted functional nature of RNA stems from its ability to form secondary structures. Therefore, this work establishes a computational method to characterize RNA secondary structure by integrating SHAPE-seq and long-read sequencing to enhance further our understanding of RNA structure in modulating the post-transcriptional regulatory processes and deciphering the influence at several layers of biological features, ranging from structure composition to consequent protein occupancy. This study will potentially impact the research community by providing methods, web interfaces, and computational pipelines, improving our functional understanding of long non-coding RNAs. This work also provides novel integration methods of technologies like Oxford Nanopore-based long-read sequencing, RNA structure-probing methods, and machine learning. The approaches developed in this dissertation are scalable and adaptable to investigate further the functional and regulatory role of RNA and its structure. Overall, this study accelerates the development of RNA-based diagnostics and the identification of therapeutic targets in human disease.

Cancer is the second leading cause of death in females. According to the American Cancer Society, there are 327,660 new cases in breast and gynecological cancers estimated in 2014, placing emphasis on the need for cancer prevention and new cancer treatment strategies. One important approach to cancer prevention involves phytochemicals, biologically active compounds derived from plants. A variety of studies on the impact of dietary compounds found in cruciferous vegetables, green tea, and spices like curry and black pepper have revealed epigenetic changes in female cancers. Thus, an important emerging topic comprises epigenetic changes due to the modulation of noncoding RNA levels. Since it has been shown that non-coding RNAs such as microRNAs and long non-coding RNAs are aberrantly expressed in cancer, and furthermore are linked to distinct cancer phenotypes, understanding the effects of dietary compounds and supplements on the epigenetic modulator non-coding RNA is of great interest. This article reviews the current findings on nutrition-induced changes in breast and gynecological cancers at the non-coding RNA level.

Huntington Disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the exon1 of huntingtin gene (HTT). The mutant HTT affects the transcriptional profile of neurons by disrupting the activities of transcriptional machinery and alters expression of many genes. In this study, we identified dysregulated non-coding RNAs (ncRNAs) in medium spiny neurons of 4-week-old HD model mouse. Also, we observed the intracellular localizations of Abhd11os and Neat1 ncRNAs by ViewRNA in situ hybridization, which could provide more precise detection, suggesting that it is a useful method to investigate the expression changes of genes with low expression levels. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University