Translational control assessed using the tethered function assay in Xenopus oocytes.
The tethered function assay is a method designed to address the role of an RNA-binding protein upon the metabolism of a reporter RNA. The basis of this assay is to artificially tether a test protein to a reporter mRNA by employing an unrelated bacteriophage MS2 or lambda N RNA-protein interaction, and to assess the effects of the test protein on the reporter RNA. In this chapter, we first discuss the principles and validity of the tethered function approach, drawing on appropriate examples from several cell types and of many proteins that regulate RNA in a variety of processes, including RNA processing (splicing, polyadenylation/deadenylation, decay), localisation and protein synthesis. Secondly, we will focus on the use of this approach to monitor translational activation and repression in Xenopus oocytes, giving a detailed protocol, and discussing possible optimizations we have explored.
Minshall
Nicola
N
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, UK.
Allison
Rachel
R
Marnef
Aline
A
Wilczynska
Anna
A
Standart
Nancy
N
D020413:3' Untranslated Regions; D000818:Animals; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D008957:Models, Genetic; D009154:Mutation; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D016366:Open Reading Frames; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D012313:RNA