Section I. Non-coding RNAs: Form, Function and Diversity

1. Genome-wide Analysis of Sense-antisense Transcripts

Chihiro Kohama and Hidenori Kiyosawa

Genomic DNA and full-length cDNA sequencing projects have provided fundamental data that have led us to the novel notion that antisense transcription is a universal phenomenon in many organisms, including mammals and plants. Successive expression analyses utilizing microarrays, genome-tiling arrays, and recent RNA sequencing with next-generation sequencers have supported this concept. We review how these genome-wide, natural antisense transcript analyses have proceeded, and we then introduce representative examples of the known functions and functional implications of antisense transcripts. The variety of modes in which antisense transcripts function in cells reveals that these transcripts do not constitute a uniform group, but perform various types of gene expression regulation.

2. Processing and Regulatory Impact of Endogenous siRNAs in Animals

Andreas Werner

The complexity of an organism is driven by a positive balance between creative and destructive forces in the process of evolution. Small non-protein-coding RNAs play an instrumental role in both regulating gene expression (creative influence) as well as suppressing selfish genetic elements (defensive role). There are three main groups of small RNAs including microRNAs, endogenous siRNAs and piRNAs. MicroRNAs represent the most important gene regulatory small RNAs and act by tuning the expression level of eukaryotic mRNAs. Interestingly, endogenous siRNAs as well as piRNAs apparently serve both regulatory and defensive purposes. They suppress the expression of repetitive DNA elements but also influence the expression of protein coding genes. For endo-siRNAs, intriguing roles in epigenetic regulation are emerging. The multiple tasks of small RNAs are in line with a role as drivers of organismal complexity.

3. Imprinted Small Non-coding RNA Genes: Time to Decipher their Physiological Functions

Stéphane Labialle, Patrice Vitali, and Jérôme Cavaillé

Genomic imprinting is a developmentally controlled form of epigenetic regulation that triggers parent-of-origin specific expression of a few mammalian genes. That is, for a given gene, only one of the two parental alleles is transcriptionnally active. Over the last several years, many imprinted small regulatory non-coding RNAs (ncRNAs, including microRNAs and box C/D small nucleolar RNAs) have been described at four evolutionarily distinct imprinted loci: the Snurf-Snrpn/Prader-Willi Syndrome and Dlk1-Dio3 chromosomal domains, and more recently at the C19MC locus and the Sfmbt2 gene. Remarkably, many imprinted ncRNA loci are clustered within large arrays formed by tandemly-repeated genes of related sequences and processed from long non-coding transcripts extending over hundreds of kilobases. Imprinted gene loci therefore give rise to unique opportunities to address both the impact of small and long non-coding RNA genes on the evolution, expression and function of mammalian genomes. In this chapter, we survey our current understanding of the functions of imprinted ncRNAs, with a particular attention to their suspected involvement in the Prader-Willi disease and higher-brain functions, as well as to their hypothetical contribution in the evolution and/or control of genomic imprinting.

Section II. Non-coding RNAs: Gene Regulation and Epigenetics

4. The X-chromosome Archetype for Noncoding RNA Regulation of the Epigenome

Daniel H. Kim and Jeannie T. Lee

X-chromosome inactivation provides a model for discovering the still emerging functions of noncoding RNAs, which exhibit diverse roles in regulating the epigenome. Combining recent insights into X-chromosome noncoding RNAs with the advent of next-generation sequencing technologies promises many new discoveries in interrogating the functions of noncoding RNAs at the genomic level. Global RNA-protein interaction information is readily available through utilizing RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq), which has recently uncovered the diversity of the noncoding Polycomb transcriptome. In this chapter, we discuss noncoding RNAs of the X-inactivation center that have provided unique insights into RNA molecules as molecular switches, guides and tethers to the epigenome, and key participants in diverse regulatory pathways, including RNA interference and Polycomb silencing.

5. Roles of Non-coding RNAs in the Control of the Coupling Between Transcription and Alternative Splicing

Mariano Alló and Alberto R. Kornblihtt

Since the discovery of splicing in 1977, alternative splicing was seen for more than two decades as an interesting mechanism to generate protein diversity but with limited genome-wide influence because it was thought to affect only 20% of mammalian genes. The sequencing of the human and other mammals' genomes in the early 2000's together with more recent high throughput analyses of splicing isoforms generated a renewed interest in alternative splicing. We know now that alternative splicing affects more than 90% of human genes, that normal and pathological cell differentiation not only depends on differential gene transcription but also on alternative splicing patterns, that mutations that either create or abolish alternative splicing regulatory sequences, named splicing enhancers and silencers, are a widespread source of human disease and that alternative splicing factors can be misregulated in cancer. The relationship between splicing and non-coding RNAs has emerged recently in the middle of an avalanche of papers showing how chromatin context could affect splicing choices. The convergence of these previously unrelated areas (non-coding RNAs, chromatin and splicing) has presented a novel and intriguing scenario that will be covered in this chapter, starting with a brief overview of transcription and splicing introducing the understanding of how these processes could be modulated by external factors. Then we will focus on our work using non-coding small RNAs (ncsRNAs) to regulate alternative splicing in human cells. Finally, we will discuss the evidence supporting the potential activity of endogenous non-coding RNAs as modulators of alternative splicing.

6. Natural Antisense Transcripts within Pseudogenes: an EST Survey

Enrique M. Muro and Miguel A. Andrade

Pseudogenes are genome loci that look like genes but have sequences apparently prevented to produce any functional product due to genetic defects. However, recent advances in the field of molecular biology urge the revisiting of this definition. In this chapter we will discuss some of those advances. There is experimental and computational evidence of some biological function arising from pseudogene transcription but this evidence is not easy to find. Accordingly, not that many studies have been published on the topic. It seems that if there is pseudogene transcript functionality, it arises in certain tissues, and in certain conditions, with much more specificity than gene expression. Some of this complexity relates to the fact that this function involves non-coding RNAs (ncRNAs), a molecular entity for which novel tools and biological paradigms are still being worked out. A particular type of ncRNAs are Natural Antisense Transcripts (NATs) and these have a special relevance for the study of pseudogene functionality for reasons that we will discuss.

7. Non-coding RNAs, Epigenomics and Complexity in Human Cells

Fabricio F. Costa

During the past two decades, new technologies in molecular biology and human genetics have enabled the discovery of different types of non-coding RNAs. Non-coding RNAs are RNA transcripts that have no apparent protein product. These molecules have been grouped in different classes such as microRNAs, small RNAs and long RNAs (lncRNAs) according to their size and function. LncRNAs have been strongly associated to epigenetic mechanisms in different cell types. Conceivably, they have been described as an essential part of the human epigenome. In this chapter, examples of lncRNAs and their function will be presented. A historical perspective on the impact of lncRNAs in epigenetic mechanisms, human disease and evolution will be also discussed.

8. Epigenetic Regulation and var Gene Expression in the Malaria Parasite Plasmodium Falciparum

Dacia Kwiatkowski and Kirk Deitsch

Much of what is currently know about complex cellular questions, including regulation of gene expression, RNA production and epigenetic influence, has been derived from experiments performed in well studied model organisms such as yeast, Drosophila, mice or human cell lines. In all of these systems, examples have been described of how non-coding RNAs (ncRNAs), which often act in accordance with epigenetic mechanisms, play a vital role in regulating gene expression. These studies have established a strong precedent for the vital role that these molecules can play in complex cellular functions, which is now being extended to other diverse organisms such as viruses and protozoans of distant evolutionary lineages. The focus of this chapter is the role that long ncRNAs play in gene regulation in the protozoan parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. In particular, we will consider ncRNAs that influence the expression of the genes encoding the primary virulence factors expressed by these parasites, the epigenetic marks associated with them, and how this process enables the parasites to avoid the immune response of their human hosts.

Section III. Non-coding RNAs: Disease and Therapeutics

9. Long Non-coding RNAs (lncRNAs) and Cancer

Jessica M. Silva and David I. Smith

There are numerous non-coding transcripts in addition to those housekeeping transcripts (ribosomal RNAs and transfer RNAs) that participate in the processes of protein production within the cells. These include the large number of small RNAs such as microRNAs, piwiRNAs and snoRNAs. In addition to these relatively small transcripts there are also considerably longer non-coding transcripts that also play important roles within the genome. These long non-coding RNAs (lncRNAs) can be differentiated from each other based upon where they are derived from within the genome. For instance, there are intronic lncRNAs (transcribed between exons of genes), intergenic lncRNAs (transcribed in the space between genes), and lncRNAs that are more complex as they overlap both intron and exon of a coding gene. Each of these lncRNAs may also be in the sense or in the antisense direction. The list of long non-coding RNAs (lncRNAs) that are associated with diseases is rapidly increasing. Some lncRNAs have been found to be aberrantly expressed in various diseases including psoriasis, mental disorders, autism, and also cancer. The list of lncRNAs associated with cancer is increasing. However, relatively little is known about the precise functions of most cancer associated lncRNAs, but of what is known suggest that these long non-coding transcripts function in many different ways within cells and promote cancer development and progression. We will review what is known about the role that some of the lncRNAs play in cancer

10. Antisense RNAs and Modulation of Tumor Suppressor Genes

Hengmi Cui, Isabelle Cui and Xi Yang

Antisense RNA is the first RNA molecule identified to function as a regulator in the cell. With advances in biological science, antisense RNA is now recognized to be involved in not only post-transcriptional regulation, but also the transcriptional regulation of various important genes including tumor suppressor genes (TSGs). Recent studies indicate that the modulation of TSGs by antisense RNA may be through either up- or down- regulation, occurring either transcriptionally, post-transcriptionally or both, dependent on features of sense and antisense. Antisense RNA is the main contributor of TSG epigenetic silencing in tumorigenesis. While a general picture of the pathways involved in antisense RNA mediated gene regulation has emerged, many questions remain unaddressed: Why does some antisense RNAs function as down-regulators but others as up-regulators in different TSGs? Are the molecular mechanisms of antisense RNA regulation highly gene- and species-specific? Are they spatially and temporally restricted? Is there an antisense RNA-induced silencing complex (ARISC)? What factor(s) cause aberrant expression of antisense RNA in tumor cells? There is the potential for using antisense RNA as a biomarker for the early diagnosis of tumors and developing new therapeutic strategies for tumor treatment by targeting and controlling antisense RNA.

11. ncRNAs in p53 Regulation

Salah Mahmoudi, Anna Vilborg and Marianne Farnebo

The p53 tumor suppressor is arguably the most important player in preventing tumor formation and progression. p53 mutations are found with high frequency (about 50%) in human tumors, however virtually all tumors have inactivated the p53 pathway in some fashion.The p53 protein functions as a transcription factor with a crucial role in orchestrating the cellular stress response. p53 can be activated by a large variety of stress factors, and reacts by triggering an appropriate response, the nature of which will vary with the triggering factor and the cellular background. The most classical outcomes of p53 activation are cell cycle arrest and apoptosis, and it is generally believed that these two, together with senescence, irreversible cell cycle arrest, are the p53 responses with greatest impact on preventing tumor formation2. However, it has lately become evident that p53 is involved in many other processes in addition to these. p53 can promote several forms of DNA repair, thus contributing to maintaining genomic integrity. Further, p53 may promote differentiation of stem and progenitor cells into more specialized cell types, and can also prevent self-renewal of stem cells. Paradoxically, p53 can also induce genes involved in promoting survival. One of these genes is p21, while being a classical cell cycle arrest-inducing p53 target, p21 antagonizes apoptosis. The list of pro-survival p53 targets also includes several antioxidant genes and genes involved in metabolism. The logic behind this surprising p53 function may be that the elimination of every cell ever exposed to any kind of stress is not desirable, while protecting us from getting cancer, it would lead to tissue degeneration. In addition to its crucial role in cancer, p53 has been implicated in several other diseases, generally diseases connected to excessive cell death. These include diabetes, cell death after ischemia, and various neurodegenerative diseases such as Huntington, Parkinson, and Alzheimer. Since p53 is able to eliminate cells through apoptosis and senescence and to induce differentiation, thus reducing stem cell populations, p53 has also been suggested to promote aging. Initial studies in mice expressing constitutively active p53 also seemed to confirm this hypothesis, However, subsequent mice models carrying an extra copy of p53 but under control of its normal regulatory elements demonstrated that properly controlled p53 did not induce aging. Instead it actually promoted longevity, largely by preventing tumorigenesis. Due to its critical function in deciding on life or death for the cell, strict regulation of p53 levels and activity is crucial. p53 protein levels are kept under tight control by multiple mechanisms. Further, p53 mRNA stability and translation is subject to regulation by a number of factors, including long non-coding RNA.

12. Natural Antisense Transcripts Mediate Discordant and Corcodant Regulation of Gene Expression

Mohammad Ali Faghihi and Claes Wahlestedt

One prominent and complex class of regulatory RNAs is natural antisense transcripts. Natural antisense transcripts (also known as antisense RNAs or antisense transcripts) are RNA molecules that are transcribed from the opposite DNA strand and are often overlapping in part with mRNA of conventional sense genes. Transcription of antisense RNA, similar to sense RNA, is tissue and cell line specific. Indeed a large fraction of antisense transcripts is expressed in specific regions of the brain, supporting involvement of these regulatory RNAs in sophisticated brain functions as well as in complex neurological disorders. Recent research on natural antisense transcripts, including several large-scale expression-profiling studies, has conclusively established the existence of antisense transcripts in eukaryotic genomes. In fact, the consensus opinion is that natural antisense transcripts, most of which represent non-protein-coding RNAs, transcribed abundantly in the mammalian genome. However, there are many unanswered questions that still exist concerning antisense transcripts biological functions and their heterogeneous mode of actions in various cells. For instance, what fraction of antisense RNAs may have functional significance, and how many different regulatory mechanisms may exist for these RNA molecules? Natural antisense transcripts appear to be utilizing various cellular pathways, but it is still not clear which intrinsic properties of antisense RNA molecules or extrinsic features, such as protein interactions, cellular and developmental context are decisive for the selection of any given pathway. How is the expression of these non-protein-coding RNAs regulated in various cells, and what are the extrinsic factors that affect the transcription of antisense RNAs? Considering tissue- and cell type-specific expression patterns of antisense RNAs and their heterogeneous proposed functions, natural antisense transcripts appear to be a heterogeneous group of regulatory RNAs with a wide variety of biological roles.

13. Exploring the Genomic Dark Matter: Non-coding RNAs and Epigenetic Regulation of Transcription as a New Therapeutic Platform

Kevin V. Morris

A growing body of evidence is beginning to emerge in human cells suggesting that particular species of non-coding RNAs can regulate gene transcription by modulating loci specific epigenetic states. While such observations were in the past relegated to imprinted genes, it is now becoming apparent that several genes in differentiated cells may be under some form of non-coding RNA based transcriptional and epigenetic control. Importantly, this form of regulation may be highly influenced by selective pressures and function in the governance and adaptability of the cell. Many studies have been carried out to date, which have begun to discern the mechanism of action whereby non-coding RNAs modulate gene transcription. Some evidence points to a role of long non-coding RNAs in controlling gene transcription by actively recruiting epigenetic silencing complexes to homology containing loci in the genome. While other studies point to a role for long intergenic non-coding RNAs in scaffold like features that are most likely equally implicit in the regulation of gene expression. The results of these studies will be considered in detail as well as the implications that a vast array of non-coding RNA based regulatory networks may be operative in human cells. Knowledge of this emerging RNA based epigenetic regulatory network has implications in cellular evolution as well as an entirely new area of pharmacopeia, namely RNA mediated epigenetic regulation of gene expression.