Detection of protein-RNA complexes in Xenopus oocytes.
There is a remarkable variety of mechanisms for controlling post-transcriptional gene expression that is achieved through the formation of ribonucleoprotein (RNP) complexes on specific cis-acting regions of mRNA. These complexes regulate splicing, nuclear and cytoplasmic polyadenylation, stability, localization, and translation. Thus, it is important to be able to detect the association of specific proteins with specific RNAs within the context of these RNP complexes. We describe a method to test for protein-RNA complexes in Xenopus oocytes. The procedure combines immunoprecipitation with reverse transcription-PCR (RT-PCR) and does not entail chemical or photo crosslinking. Microinjected mRNA is efficiently translated in Xenopus oocytes; thus, in cases where primary antibody is not available, an epitope-tagged version of the protein can be expressed for utilization in this procedure. The inclusion of control mRNAs has provided no evidence of nonspecific protein reassociation to RNA during or subsequent to cell lysis. The method has been used to document the association of certain trans-acting factors specifically with localized mRNAs in Xenopus oocytes.
Huber
Paul W
PW
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. huber.1@nd.edu
Zhao
Wei-meng
WM
D000818:Animals; D003584:Cytological Techniques; D000939:Epitopes / Q000737:chemistry; D047468:Immunoprecipitation; D009865:Oocytes / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry; D014981:Xenopus / Q000378:metabolism*
D000939:Epitopes; D011506:Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D012313:RNA; D012685:Sepharose