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In recent years, small regulatory RNAs have been discovered at a staggering rate both in prokaryotes and eukaryotes. By now it is clear that post-transcriptional regulation of gene expression mediated by such RNAs is the rule rather than—as previously believed—the exception. In this chapter, we focus on small RNAs (sRNAs) encoded by bacterial chromosomes. The strategies for their discovery, their biological roles, and their mechanisms of action are discussed. Even though the number of well-characterized sRNAs in, for example, the best studied model enterobacterium Escherichia coli , is still small, the emerging pattern suggests that antisense mechanisms predominate. In terms of their roles in bacterial physiology, most of these RNAs appear to be involved in stress response regulation. Some other examples indicate functions in regulation of virulence. Two aspects of sRNA-mediated control arising from recent observations are addressed as well. Firstly, some sRNAs need proteins (notably Hfq) as helpers in their antisense activities—at this point the reason for this requirement is not understood. Secondly, only limited sequence complementarity is generally observed in antisense–target RNA pairs. This raises the fundamental question of how specific recognition is accomplished, and what the structure/sequence determinants for rapid and productive interaction are.

The revelation in the last few years of a large number of new noncoding RNAs (ncRNAs) has revolutionized our view of how gene regulation works. This is to a large extent due to the recent advances in computational as well as experimental methodology, combined with an increasing number of sequenced genomes which have had an enormous impact on the quest for new small ncRNAs. These RNAs have a function per se and are not merely intermediates in the transfer of information from genes to proteins. Instead they have turned out to be involved in the regulation of many complex biological processes, including stress response, cell differentiation, and even in control of diseases. This chapter briefly describes some of the methods used to identify and isolate different classes of ncRNAs in the size range 50–500 nt. One of these, small nucleolar RNAs, will be discussed in detail.

Over the last few years several specialized software tools have been developed, each allowing a certain class of RNAs insequencedatatobe found.Herewedescribeageneral tool that allows us to specify many different non-coding RNAs and structural RNA elements by a simple pattern description language.To take into account that RNA is normally conserved in structure as well as in sequence, the pattern description language combines methods to describe sequence and structural similarities as well as further characteristics, e.g., thermodynamic constraints. Structure- and sequence-based patterns describing certain classes of RNAs are collected in a web-based pattern library. These include simple patterns, e.g., describing extrastable tetraloops and small regulatory stem-loop structures, as well as more complex patterns, for example describing pseudoknots, ribozymes, SRP RNAs, 5S RNA and selenocysteine insertion sequences.Aweb-based service allows a user to search the patterns fromthe library in sequences given by the user. Alternatively, the user can specify a pattern that is searched for in public genomic sequence data. Here we give a comprehensive introduction of the pattern language, describe how to systematically derive pattern descriptions, and show some results on purine riboswitches obtained using this computational approach.

Micro-RNAs (miRNAs) are single-stranded RNA molecules of about 20 nucleotides and represent a class of small non-coding regulatory RNAs found in higher eukaryotes across kingdoms. miRNAs originate from endogenous chromosomal genes that encode a transcript, called pre-miRNA, of about 70 nucleotides in length. Several premiRNAs can be combined in a polycistronic pri-miRNA. Both precursors are processed by specific ribonucleases: Drosha cleaves a pri-miRNA to several pre-miRNAs, which are processed by a Dicer nuclease into the mature miRNA. The miRNAs themselves interfere with gene expression by base-pairing with a messenger RNA (mRNA) target, but one miRNA can bind in a specific manner to several mRNAs; therefore, miRNAs form a regulatory network that controls gene expression. In this way they are believed to determine tissue-specific gene expression and to act as checkpoints for developmentally important processes. Only a few plant miRNAs show a perfect match to their mRNA target, but the majority of miRNAs, including mammalian miRNAs, regulate translation by imperfect base-pairing. Each species is expected to have a couple of hundred miRNAs and only a few validated targets are known so far. Several attempts have been made to identify miRNA targets by bioinformatics.

Double-stranded RNA has long been known to be a trigger for cellular responses to viral infections, leading to dramatic changes in cellular processes. Since the advent of RNA interference, it has become clear that double-stranded RNA also causes specific effects, regulating gene expression on the transcriptional, post-transcriptional and translational levels. An essential prerequisite for double-stranded RNA effects is proteins that specifically recognise these molecules in order to elicit the cellular response. This chapter focuses on the function and molecular architecture of those proteins that interact with double-stranded RNA and that are key players in the RNA interference, editing and the PKR response. After summarising the origin of double-stranded RNA molecules and structural features of A-type helices, the way proteins can interact with this secondary structure is discussed. The variability of domain structures of proteins that are functional homologues in processes triggered by double-stranded RNA is reviewed and consequences resulting from the different design of proteins from various organisms are discussed. Finally, differences and similarities of pathways with respect to their subcellular localisation and the length of the double-stranded RNA trigger are summarised.

RNA silencing is a conserved regulatory mechanism that plays an important role in genome integrity and defense in eukaryotic organisms. A key molecule in this sequence-specific RNA degradation mechanism is double-stranded RNA, which is processed by an RNase-III like enzyme (Dicer) into small interfering RNAs (siRNAs). The initial pool of siRNAs can be amplified through the action of RNA-dependent RNA polymerases, which could account for the observed spreading of RNA silencing along the target gene (transitive silencing) and throughout the organism (systemic silencing). In this chapter we discuss the mechanism of RNA amplification and its possible involvement in transitive and systemic RNA silencing in different organisms.

Gene silencing by RNA interference (RNAi) and by antisense RNA are powerful tools to interfere with the expression of eukryotic genes. Since the first description of RNAi in 1998, antisense-mediated gene silencing has been considered to have essentially the same mechanism as gene silencing by RNAi. However, while substantial effort has been made to dissect the RNAi pathway, the cellular machinery that is responsible for posttranscriptional regulation by antisense RNA is rather poorly defined and direct comparisons between the RNAi and antisense experiments are rare. Even though similarities are very likely, recent data suggest that in addition to the expected overlaps in the pathways, there are also mechanistic differences and different requirements for specific gene products. We will summarize the current state of knowledge of the antisense RNA and RNAi mechanisms and address some of the open questions in the field. We will further provide some evidence suggesting that gene silencing by antisense RNA and by RNAi represent related but not identical mechanisms. A model to explain the partially overlapping pathways will be presented and may contribute to the further understanding of posttranscriptional gene regulation.

Transposons are mobile genetic elements that live parasitically within the genome of cellular organisms. They can affect the fitness of their hosts by influencing gene function, gene activity, genome structure, and overall DNA content. Since excessive transposon activity can result in a high mutagenic rate and genomic instability, eukaryotes have evolved epigenetic mechanisms to reduce transposition to manageable levels. The alga Chlamydomonas reinhardtii appears to have several, at least partly independent, transposon repression pathways that operate at either the transcriptional or the post-transcriptional level. Two genes have been implicated in the transcriptional silencing of transposons and single-copy transgenes: Mut9 , which encodes a novel serine/ threonine protein kinase capable of phosphorylating histones H3 and H2A, and Mut11 , which encodes a WD40-repeat containing protein. The Mut11 protein functions as a subunit of a histone methyltransferase complex(es) that is required for monomethylation of histone H3 lysine 4 and the maintenance of repressed euchromatic domains. These mechanisms of transcriptional gene silencing operate independently from the RNA interference (RNAi) machinery.

In animals noncoding RNAs are involved in a large variety of gene silencing mechanisms. These include post-transcriptional RNA interference (RNAi) that is mediated by small double-stranded RNAs and results in degradation of messenger RNAs as well as epigenetic silencing of genes. RNAi as a naturally occurring silencing mechanism has been well investigated in various eukaryotic organisms. Sequencing of the human and mouse genomes and careful analyses of the related transcriptomes led to the identification of some hundred microRNAs that might regulate endogenous gene expression by RNAi-like mechanisms or repression of translation. In mammals the major protein components of the RNAi machinery have been identified, and RNAi has become a tool for artificial gene silencing in mammalian systems. There is also evidence that in mammalian cells genes can be regulated by noncoding antisense transcripts that are transcribed from the opposite DNA strand. Besides their potential roles in RNAi and repression of translation, short double-stranded RNAs and also long noncoding RNAs are involved in epigenetic gene silencing. In this chapter we give an overview of prominent features of naturally occurring RNAi and also of the potential role of RNAs in epigenetic gene silencing mechanisms in animals, especially in mammals.

The recent advent of genomic tools has provided us with very powerful ways for the identification of functional genes, thus enhancing our understanding of the molecular basis of both normal and disease phenotypes. Now that sequence information is available for many genomes, a simpler and more definitive technology for the rapid identification of functional genes is a current focus of interest. A simple screening system based on the catalytic activity of ribozymes, whose target specificities are coupled with loss-of-function mutants, has been developed to isolate key genes involved in a defined phenotype. The system was validated for functional gene screens including apoptosis, transformation, metastasis, and muscle and normal differentiation. This system should be applicable to the identification of functional genes involved in many cellular processes and diseases.