title: Slicer activity in Drosophila melanogaster S2 extract.
abstract: Reconstitution of RNA-inducing silencing complex (RISC) in vitro is a powerful biochemical technique to analyze crucial steps in RNA interference (RNAi) pathways. RISC contains an RNase enzyme, Argonaute, which is guided by small interfering RNA (siRNA) to recognize and silence its targets. Drosophila S2 cell extract is a good source of enzymes and factors that faithfully recapitulate essential steps in RISC assembly and function. In this chapter, we will describe how to prepare enzymatically active cell-free S2 extract to analyze the Slicer activity of RISC as well as the effects of viral RNAi suppressors which block this process.
authors: Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.
authors: Arabinda
authors: A
authors: Nayak
authors: Raul
authors: R
authors: Andino
mesh_terms: D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D002460:Cell Line; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics / Q000276:immunology / Q000821:virology; D057075:Enzyme Assays / Q000379:methods*; D016153:Genes, Suppressor; D005814:Genes, Viral / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D007304:Insect Viruses / Q000235:genetics / Q000502:physiology; D007553:Isotope Labeling; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D002457:Cell Extracts; D012333:RNA, Messenger; D006160:Guanosine Triphosphate; D016914:Ribonuclease H