1. The Hammerhead Ribozyme Revisited: New Biological Insights for the Development of Therapeutic Agents and for Reverse Genomics Applications

Justin Hean and Marc S. Weinberg

Hammerhead ribozymes are the smallest known naturally occurring ribozymes which are capable of catalyzing the endonucleolytic trans-esterification of RNA. A recent re-examination of the catalytic properties of naturally-derived hammerhead ribozymes has resulted in a better understanding of the catalytic efficiency of this enzyme in vitro and in vivo. The minimal trans-cleaving hammerhead ribozyme has been a ubiquitous tool in both genomics and therapeutics research over the last twenty years and these new insights into hammerhead ribozyme biochemistry may offer hope for the generation of improved trans-cleaving ribozymes which function effectively in vivo. Next-generation hammerhead ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.

2. Epigenetic Regulation of Gene Expression

Kevin V. Morris

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. While chromatin remodeling and DNA methylation have been studied for several years now far less is know about how these epigenetic marks are directed to each particular gene. Recently, however the role of RNA in epigenetic gene regulation has begun to become apparent. In this chapter we will discuss the basic mechanisms of epigenetic regulation of gene expression and how epigenetics might be involved in the evolution of the cell.

3. The Role of RNAi and Noncoding RNAs in Polycomb Mediated Control of Gene Expression and Genomic Programming

Manuela Portoso and Giacomo Cavalli

Regulation of gene expression is a complex, multi-layered process that is crucial to correctly drive and maintain cell identity during development and adult life. In this chapter, we discuss the functional and molecular links between two well-conserved gene silencing pathways, RNA interference (RNAi) and Polycomb. RNAi participates in post transcriptional as well as transcriptional gene silencing of natural genes as well as transposons and viruses. Polycomb group (PcG) proteins are well-known for their role in silencing HOX genes through modulation of chromatin structure. However, both mechanisms were found to be involved in specific epigenetic processes like cosuppression in Drosophila melanogaster and the formation of C elegans mes and SOP-2 complexes. Recent work has uncovered molecular links between RNAi components and Polycomb-mediated silencing in human cells and Drosophila. RNA polymerase II and Argonaute 1 interact to bring about chromatin modifications on endogenous Polycomb target gene promoters in human cells, while Drosophila RNAi components modulate the nuclear organization of PcG target DNA elements, thereby affecting the strength of PcG-mediated silencing. Finally, we discuss the findings of several microRNAs and non coding RNAs in human and fly HOX gene loci, where they may regulate HOX gene expression both post-trascriptionally and co-transcriptionally.

4. Heterochromatin Assembly and Transcriptional Gene Silencing under the Control of Nuclear RNAi: Lessons from Fission Yeast

Aurélia Vavasseur, Leila Touat-Todeschini and André Verdel

Heterochromatin is a prevalent chromatin state among eukaryotes that has critical functions in chromosome segregation, control of genomic stability and epigenetic regulation of gene expression. Here, we review studies conducted in the fission yeast Schizosaccharomyces pombe, which reveal that two RNAi complexes, the RNAi-induced transcriptional gene silencing (RITS) complex and the RNA-directed RNA polymerase complex (RDRC), are part of a RNAi machinery involved in the initiation, propagation and maintenance of heterochromatin assembly. It appears that these two complexes localize in a siRNA-dependent manner on chromosomes, at the site of heterochromatin assembly. Moreover, these studies reveal an unprecedented and central role for RNA polymerase II (RNApII) in RNAi-dependent heterochromatin assembly. RNApII synthesizes a nascent transcript that is believed to serve as a RNA platform to recruit, RITS, RDRC and possibly other complexes required for heterochromatin assembly. Finally, recent findings indicate that RNAi as well as an exosome-dependent RNA degradation process contribute to heterochromatic gene silencing. These findings challenge the widely accepted view that heterochromatic gene silencing is caused strictly by chromatin compaction. As RNAi-dependent chromatin modifications have been observed throughout the eukaryotic kingdom the mechanisms reviewed here are susceptible to occur in a large range of eukaryotes.

5. RNA-Mediated Gene Regulation in Drosophila

Harsh H. Kavi, Harvey R. Fernandez, Weiwu Xie and James A. Birchler

Short RNAs are increasingly recognized to play multiple roles in affecting gene expression at many levels as illustrated by work in Drosophila. Here we review the biochemical parameters of RNA interference, the technique that uses double stranded RNA, which is cleaved by Dicer to produce small interfering RNAs (siRNAs), as guides to cleave homologous mRNAs. This process is believed to occur in the cytoplasm and is used in the endogenous process of viral resistance. In addition, many of the same gene products are also involved in transcriptional gene silencing processes. This was first documented for cosuppression of white-Alcohol dehydrogenase transgenes, which is associated with the Polycomb repressive complex of chromatin proteins. Genetic studies of RNA silencing genes also implicate a role in heterochromatin silencing. Some gene products involved in RNAi are also involved in the formation of repeat associated small RNAs (rasiRNAs), whose formation appears to be Dicer independent and critical for repressing transposon expression particularly in the germline. Roles for small RNAs are also implicated in chromatin insulator activity, the integrity of the nucleolus and long-range associations of homeotic genes.

6. MicroRNA-Mediated Regulation of Gene Expression

Lena J. Chin and Frank J. Slack

MiRNAs are short, ~22 nucleotide regulatory RNAs, first discovered in Caenhorhabditis elegans. Since then, hundreds of miRNAs have been identified in plants and animals. Based on the current number of predicted miRNAs, one to three percent of genomic DNA is believed to encode these small, regulatory RNAs. MiRNAs inhibit protein synthesis by binding to their target mRNAs and regulating gene expression in a post-transcriptional manner. The exact mechanism by which target gene expression is down-regulated is unclear; however, experimental evidence has led to several different theories to explain miRNA-mediated mRNA repression. These possible mechanisms include target degradation, localization to P-bodies, inhibition of translation initiation or elongation, mRNA deadenylation, and mRNA destabilization.

7. Viral Infection-Related MicroRNAs in Viral and Host Genomic Evolution

Yoichi R. Fujii and Nitin K. Saksena

MicroRNA (miRNA) is a small RNA (~22 nucleotides). The miRNA genes have been discovered in plants, invertebrates, and vertebrates. These miRNAs can regulate gene expression to inhibit translation of target messenger RNAs (mRNAs), and sometimes direct many rounds of site-specific mRNA cleavage in mammalian cells. Beyond the cutting-edged criteria of RNA interference (RNAi) by complementarily pairing of short interfering RNA (siRNA), the miRNAs, which were incomplementarily paired, are also encoded by several viruses, such as herpesviruses and human immunodeficiency virus type 1 (HIV-1). Intriguingly, gene expression of HIV-1 genome has been recently shown to be epigenetically regulated via a novel process of transcriptional repression by the miRNA of virus itself. However, role of the viral miRNA for gene regulation is not still well understood mechanistically as compared with host miRNAs. Conversely, the experimental and computational methods used to detect and predict the miRNA target genes are common ones in genome informatics. We describe in this chapter about the target prediction of a viral miRNA, miR-N367 and the conservation of secondary structure of pre-miR-N367 into mir-98/let-7 and mir-181a-2 in human miRNAs whose targets in HIV-1 genome could be related to HIV-1 transcriptional system. Further, we show here that the sequences of viral nef si/miRNA are conserved in plant miRNAs and the nef si/miRNAs was enabled to express in Arabidopsis thaliana similar to human cell. Thus, we hypothesize that the orphaned non-selfish miRNAs may evolve and jump on to other RNAs, which can transposably lead to spread of these miRNAs from some plant and vertebrate genomes through feeding of miRNAs-containing foods, viruses, etc. Equivocally, miRNAs can be picked up into the lentiviral transposon, such as HIV-1. Therefore, the viral miR-N367 would necessarily be a HIV-1 silencer to be inclusively incorporated into HIV-1 itself. The virulence may be lost with recombination and mutation by miRNAs from the viral and host genome. The question is, whether the encoded miRNAs are mediating the viral and host genomic evolution, which remains to be answered.

8. Regulation of Mammalian Mobile DNA by RNA-Based Silencing Pathways

Harris Soifer

The field of RNA silencing has grown quickly in less than a decade from the unexpected observation of color variation in transgenic flowers to a conserved eukaryotic mechanism for regulating gene expression at the post transcriptional level. Genetic and biochemical dissection of RNA silencing processes in many eukaryotes indicate that multiple pathways operate to control the expression of target genes, often in a developmentally or tissue-specific manner. What has also become clear, however, is that specialization of RNA silencing components and trigger molecules that occurred during speciation has lead to differences in the regulation of target RNAs across species. In lower eukaryotes, such as Drosophila and C. elegans, where the study of RNA silencing processes has benefited greatly from ability to carry out targeted gene inactivation and large-scale genetic screens, the activity of mobile genetic elements within these genomes is under the direct control by one or more RNA-based silencing mechanisms. Mammalian genomes are dominated by retrotransposons, mobile genetic elements that move through an RNA intermediate and thus would seem to be obvious targets for an RNA-based silencing mechanism that relies on complementarity with the transcript. The idea that mammalian RNA silencing pathways function, in part, to regulate the activity of mobile genetic elements is supported by several empirical studies, although the mechanism of retrotransposon regulation by RNA appears to work by inducing DNA methylation at promoter sequences rather than through degradation of RNA. In this chapter, an overview of RNA silencing mechanisms in mammalian cells is presented along with an outline highlighting the different mobile genetic elements that inhabit the mouse and human genomes. A significant portion of the chapter is devoted to empirical evidence supporting a direct role for mammalian RNA silencing pathways in the regulation of mobile DNA. Furthermore, recent bioinformatic data suggesting that ancient mobile elements once regarded as "junk" have evolved into functional, non-coding RNA that serve as triggers for RNA-based gene regulation in mammalian cells are also discussed.

9. The Role of Non-Coding RNAs in Controlling Mammalian RNA Polymerase II Transcription

Stacey D. Wagner, Jennifer F. Kugel and James A. Goodrich

Controlling transcription of protein-encoding genes into mRNA is central to all basic biological processes and understanding mechanisms of transcriptional control has been the focus of intense investigation. In the case of mammalian protein-encoding genes, a multitude of protein factors that regulate mRNA transcription have been discovered and characterized over the past four decades. During that time a few RNAs were found to play a role in transcriptional regulation in very specific biological situations. Only recently has the scientific community begun to appreciate that RNAs play a much larger part in regulating mammalian transcription. This appreciation grew out of the discoveries of a number of RNA transcriptional regulators, some of which have the potential to control entire transcriptional programs in response to extracellular stimuli. At the same time, studies have shown that a much larger portion of the genome is transcribed than previously thought, and that many of the transcripts produced in mammalian cells do not encode protein, and hence are called non-coding RNAs (ncRNA). With increased awareness of the regulatory potential of ncRNAs, researchers have begun specifically looking for ncRNA transcriptional regulators. This search will likely result in a dramatic increase in the number and complexity of these novel transcriptional regulators over the next few years. Here, we review ncRNAs that control mammalian mRNA transcription. The ncRNAs discussed are grouped by the specific stage of transcription that they target. Intriguingly, most stages of the reaction are now known to be targeted by at least one ncRNA, from the mobilization of activators through the termination of transcription. Throughout the chapter we also include our thoughts on the pressing questions that could next be addressed to further our understanding of each of the ncRNAs.

10. Pyknons as Putative Novel and Organism-Specific Regulatory Motifs

Isidore Rigoutsos

In recent work, we introduced a new type of putative regulatory motif that we named "pyknon" from the greek adjective for dense. By definition, pyknons are variable length sequences with a statistically significant number of intact copies in the intergenic and intronic regions of the genome and additional copies in the untranslated or amino acid coding regions of known transcripts. Even though the original presentation discussed pyknons in the context of the human genome, we think that pyknons likely represent a more general architectural component of eukaryotic genomes. The exact role of pyknons is currently unclear but the findings so far support a regulatory responsibility. In this chapter, we review the process that led to this discovery, the results so far, and briefly present some thoughts about the possibility that pyknons hint at a previously unseen layer of cell process regulation.

11. RNA-Mediated Recognition of Chromosomal DNA

David R. Corey

Designed molecules that recognize specific sequences within chromosomal DNA would provide useful probes for natural cellular processes, tools for laboratory experimentation, and lead compounds for therapeutic development. We initially discovered that duplex DNA could be recognized by conjugates consisting of DNA oligonucleotides and cationic proteins or peptides. We subsequently observed similarly efficient recognition by neutral peptide nucleic acids (PNAs). Taking these studies as a starting point, we examined whether duplex RNAs could also mediated efficient recognition of duplex DNA. We found that RNAs can target transcription start sites and either inhibit or activate gene expression. These data, together with that from other laboratories, indicate that promoter-targeted RNAs can be powerful tools for regulating gene expression.

12. RNA Mediated Transcriptional Gene Silencing: Mechanism and Implications in Writing the Histone Code

Kevin V. Morris

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Exactly how these epigenetic modifications are directed to the particular gene and the local chromatin has remained enigmatic. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. Recently we and others have shown in human cells that RNA can specifically direct epigenetic modifications to targeted loci (the promoter regions) and modulate silencing. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by the antisense strand of small-interfering RNAs (siRNAs). RNA mediated transcriptional gene silencing, directed DNA methylation as well as the putative mechanism involved in the context of Human cells will be discussed. Undoubtedly, the ramifications from these recent observations represent a paradigm shift in which a hidden layer of complexity is involved in gene regualtion and is operative via the action RNA essentially epigenetically regulating DNA.

13. Small RNA-Mediated Gene Activation

Long-Cheng Li

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. Recent discoveries now suggest that dsRNAs may also act as small activating RNAs (saRNA). By targeting sequences in gene promoters, saRNAs readily induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa). This discovery expands our knowledge on both the functionality and complexity small RNAs have in regulating gene expression. In this chapter, we will examine the evidence accumulated thus far on RNA molecules that positively regulate gene expression and function.

14. Therapeutic Potential of RNA-mediated Control of Gene Expression: Options and Designs

Lisa Scherer and John J. Rossi

We review factors to consider when choosing an mRNA knockdown approach in human cells using therapeutic expressed RNAs. We emphasize methods that use RNA triggers of endogenous cellular pathways to target and degrade mRNAsÐRNAu, RNase P external guide sequences, tRNAse Z^L small guide sequences, and RNAiÐ rather than those RNAs with intrinsic activity, such as ribozymes and aptamers. The range of available methods may be particularly important in combinatorial approaches to inhibit viruses prone to mutational escapes, such as HIV-1 and HCV.