Isolation of RNP granules.
The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient "handle" for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.
Department of Clinical Biochemistry, Copenhagen University Hospital, Copenhagen, Denmark. lars.joenson@rh.regionh.dk
Lars
L
Jønson
Finn Cilius
FC
Nielsen
Jan
J
Christiansen
D015153:Blotting, Western; D003594:Cytoplasmic Granules; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics*; D047468:Immunoprecipitation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism; D013752:Tetracycline
C406144:IGF2BP1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D013752:Tetracycline