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.

The study of RNA interference, editing, and modification has led to major shifts in our understanding of how genes are expressed and regulated. In RNA Interference, Editing, and Modification: Methods and Protocols, hands-on experimentalists describe in detail the protocols and assays they have developed to study these processes in most of the major biological systems in which they are known to occur-a wide range of organisms that includes worms, flies, trypanosomes, mammals, and plants. A historical overview of each subject is provided to put related chapters into a larger context. RNA interference (RNAi) has also emerged in recent years as an important and promising tool for gene discovery and reverse genetic engineering. Application of this cutting-edge technique is described for a range of experimental systems. Topics of interest include stable and transient RNA interference, RNA editing, gene silencing, bioinformatics, small noncoding RNAs, and RNomics. Special attention is given to methods for the identification and characterization of small RNAs, many of which are involved in RNA interference or modification. In addition, techniques used to study RNA editing mechanisms are elaborated for both systems that involve the conversion of one base to another and insertion-deletion editing. To promote the adaptation of their assays and approaches for use in other biological systems, the authors have included a range of methods representative of the major experimental systems in a given field. All protocols presented follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
State-of-the-art and highly practical, RNA Interference, Editing, and Modification: Methods and Protocols offers not only geneticists, molecular biologists, andbioinformaticists a comprehensive collection of readily reproducible techniques for the elucidation of gene function and the alteration of gene expression, but also describes how best they may be productively used to address outstanding mechanistic questions and adapted to novel biological systems.