The diversity of RNAi and its applications

Inhibiting the expression of genetic information has long been the domain of classical genetics. In recent years, some useful nucleic acid-based tools have been developed that allow studies of gene function in organisms that are not amenable to rapid genetic analyses. These tools include antisense oligomers, ribozymes, DNAzymes, and aptamers. Many useful analyses have been conducted with these reagents, and in some instances the reagents have been developed into therapeutics. Most recently, the discovery of an endogenous mechanism for regulation of gene expression, RNA interference (RNAi), has created a small revolution in functional genomics, which is rapidly spreading into therapeutics. RNAi is a highly conserved phenomenon in eukaryotes, apparently evolved as a defense mechanism against viral infections. Although RNAi was observed in plants more than a decade ago, it is triggered by short (<25 nucleotides) double-stranded duplexes processed from longer RNA duplexes. There are two major classes of these small RNAs, the microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs most often derive from longer precursor hairpin transcripts whose stems form imperfect Watson-Crick helices. The siRNAs are derived from double-stranded RNA (dsRNA) precursors with complete Watson-Crick base pairing, most often viral in origin. Both of these RNAs assemble with a core of proteins called the RNA-induced silencing complex, or RISC. The most prominent members of RISC are a family of proteins called Argonaute. One member of this family, Argonaute 2, has a catalytic domain that functions to cleave one strand of a duplex RNA, resulting in functional destruction of the cleaved strand. Other members of this family lack the catalytic domain and thus function by recruiting proteins that inhibit translation, possibly at the step of initiation. Functionally, miRNAs and siRNAs inhibit gene expression by different mechanisms. miRNAs most often bind to the 3′ untranslated region of target transcripts, forming incomplete Watson-Crick base pairs and blocking translation. siRNAs form perfect or near perfect Watson-Crick helices anywhere along a target mRNA and direct sequence-specific cleavage of the target. It is important to note that miRNAs and siRNAs are interchangeable, depending upon the extent of base pairing with the target mRNA. A third function of small RNAs is the silencing of gene expression at the level of transcription by signaling the formation of heterochromatin, termed transcriptional gene silencing (TGS). This mechanism is prominent in fission yeast, where it is involved in maintaining a heterochromatic state in centromeric regions, and in plants, in which TGS plays a prominent role as an anti-viral defense mechanism. Recent evidence has shown that TGS can be induced in mammalian cells, although its role in mammalian gene regulation is presently unknown. This special supplement covers a wide variety of topics ranging from therapeutic potential of RNAi in combating human diseases to the role of RNAi in TGS and in suppressing diseaselinked messenger RNA (mRNA) isoforms. In the following section, we provide a brief introduction to each of the reviews. This supplement starts with a review by Kevin Morris that describes advances in our understanding of RNAi-mediated TGS. TGS was first observed in tobacco plants, and subsequent studies showed that dsRNAs direct methylation of histones, resulting in a series of chromatin structural alterations, which ultimately lead to chromatin silencing. Recent studies confirmed that dsRNA-mediated TGS is present in Arabidopsis, Schizosaccharomyces pombe, and mammalian cells. The fact that siRNA can mediate gene silencing at the level of transcription has opened up new avenues in controlling gene expression by directing epigenetic changes in local chromatin structure. INTRODUCTION