Analysis of Double-Stranded RNA and Small RNAs Involved in RNA-Mediated Transcriptional Gene Silencing

RNA-mediated gene silencing is triggered by double-stranded RNAs (dsRNAs) that are processed to short RNAs approx 21–25 nucleotides in length. The short RNAs act as guides for enzyme complexes that degrade, modify, or inhibit the function of homologous target nucleic acids. There are three general types of RNA-mediated silencing: (1) RNA interference (RNAi) and the related processes of posttranscriptional gene silencing in plants and quelling in Neurospora crassa , which involve sequence-specific mRNA degradation; (2) microRNA-guided repression of translation; and (3) RNA-directed epigenetic modifications of the genome, such as DNA cytosine (C) methylation and histone methylation (1 ). This chapter focuses on a specific aspect of the third type of silencing: RNA-directed promoter methylation and transcriptional gene silencing (TGS).