Ribotrap : targeted purification of RNA-specific RNPs from cell lysates through immunoaffinity precipitation to identify regulatory proteins and RNAs.
Many elegant methodologies have been devised to explore RNA-protein as well as RNA-RNA interactions. Although the characterization of messages targeted by a specific RNA-binding protein (RBP) has been accelerated by the application of microarray technologies, reliable methods to describe the endogenous assembly of ribonucleoproteins (RNPs) are needed. However, this approach requires the targeted purification of a select mRNA under conditions favorable for the copurification of associated factors including RNA and protein components of the RNP. This chapter describes previous methods used to characterize RNPs in the context of in vitro approaches and presents the Ribotrap methodology, an in vivo protocol for message-specific purification of a target RNP. The method was developed in a yeast model system, yet is amenable to other in vivo cell systems including mammalian cell culture.
Beach
Dale L
DL
University of North Carolina, Chapel Hill, USA.
Keene
Jack D
JD
D020413:3' Untranslated Regions; D000345:Affinity Labels; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D047468:Immunoprecipitation / Q000379:methods*; D008967:Molecular Biology / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D020413:3' Untranslated Regions; D000345:Affinity Labels; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA