Using the lambdaN peptide to tether proteins to RNAs.
Proteins interacting with messenger RNAs (mRNAs) affect their nuclear processing, export, translation efficiency, stability, or cytoplasmic localization. Such RNA-binding proteins are often modular, containing RNA-binding domain(s) and other functional modules. To analyze the function of such proteins independent of their normal RNA-binding domains or to introduce effector modules to defined RNA-binding regions, a number of tethering approaches have been developed, often based on the use of large proteins and their specifically interacting RNA sequences. Here we report the use of a versatile system to tether proteins to mRNAs. The 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (lambdaN-(1-22) or lambdaN peptide) is used to tag the protein of interest, and its specific 19 nt binding site (boxB) is inserted into the target RNA recruiting the properties of the fusion protein to the RNA. The major advantage of this system derives from the small size of the peptide and its target sequence, which facilitates cloning and its use for biochemical experiments and diminishes possible interferences with the fused protein. The chapter illustrates the use of this system to create dedicated mRNA-specific factors involved in processes, such as mRNA translation and nonsense-mediated mRNA decay.
Baron-Benhamou
Julie
J
Rockefeller University, New York, NY, USA.
Gehring
Niels H
NH
Kulozik
Andreas E
AE
Hentze
Matthias W
MW
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; C111931:ELF4 protein, human; C059417:N protein, Bacteriophage lambda; D010446:Peptide Fragments; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C435488:UPF3B protein, human; D054334:Viral Regulatory and Accessory Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase