RNA Interference, Editing, and Modification: Methods and Protocols

Two of the more fascinating biological phenomena that have been d- covered in recent years are RNA editing and RNA interference. Each of these processes has been found in a cross-section of biological systems, including mammals, viruses, plants, and a range of model organisms (C. elegans,Dro- phila, and various lower eukaryotes). RNA editing, which results in an RNA product different from that predicted by the genome, occurs through a variety of mechanisms. Alterations can occur at either the base level, in which one base is changed to another (substitutional editing/base modification), or via the addition and/or deletion of nucleotides relative to the original template (insertion/deletion editing). RNA interference (RNAi) involves the specific degradation of targeted mRNAs. Although RNA interference, editing, and modification use different enzymes and mechanisms, the targets of each of these reactions are often specified by RNA molecules. Indeed, the discovery of guide RNAs (gRNAs) that direct nucleotide insertion and deletion in trypa- some mitochondria set the precedent for subsequent discoveries of the small nuclear RNAs (snoRNAs) that target pseudouridylylation and methylation of stable RNAs and the small double-stranded RNA fragments (siRNAs) that mediate RNAi. Other small RNAs are known to mediate translational regu- tion during development (small temporal RNAs [stRNAs]) and mRNA stab- ity (microRNAs [miRNAs]), and the recent identification of more than a hundred small "noncoding" RNAs has led to the realization that they may represent only the proverbial "tip of the iceberg.

Table of Contents:
RNA Interference and Gene Silencing.- RNA Interference.- Methods for Delivery of Double-Stranded RNA into Caenorhabditis elegans.- Induction and Biochemical Purification of RNA-Induced Silencing Complex From Drosophila S2 Cells.- Analysis of Gene Function in Trypanosoma brucei Using RNA Interference.- Short Hairpin Activated Gene Silencing in Mammalian Cells.- Geminivirus Vectors for Transient Gene Silencing in Plants.- Posttranscriptional Gene Silencing in Plants.- Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning.- RNA Editing and Modification.- A Historical Perspective on RNA Editing.- Identification of Substrates for Adenosine Deaminases That Act on RNA.- Purification and Assay of Recombinant ADAR Proteins Expressed in the Yeast Pichia pastoris or in Escherichia coli.- Isolation of an mRNA-Binding Protein Involved in C-to-U Editing.- In Vitro Assays for Kinetoplastid U Insertion-Deletion Editing and Associated Activities.- Identification and Characterization of Trypanosome RNA-Editing Complex Components.- Chimeric Templates and Assays Used to Study Physarum Cotranscriptional Insertional Editing In Vitro.- Methods for Analysis of Mitochondrial tRNA Editing in Acanthamoeba castellanii.- In Vitro RNA Editing Systems From Higher Plant Chloroplasts.- Studying RNA Editing in Transgenic Chloroplasts of Higher Plants.- Detection and Quantification of Modified Nucleotides in RNA Using Thin-Layer Chromatography.- Functional Characterization of 2?-O-Methylation and Pseudouridylation Guide RNAs.- Experimental Rnomics.