Assaying nuclear messenger RNA export in human cells.
This chapter describes a simple method for the analysis of nuclear messenger RNA (mRNA) export in human cells in culture. The assay described relies on the observation that mRNA molecules containing an intron are generally retained in the nucleus until splicing is completed. Upon sequestration of the cat indicator gene in a single intron located 5' to an mRNA cap site, CAT protein expression becomes dependent on the specific recruitment of a nuclear RNA export factor to the unspliced cat RNA via an inserted RNA binding site. This site can be a natural, high affinity RNA target for the nuclear export factor or alternately the export factor can be tethered to the unspliced cat mRNA by fusion to a heterologous RNA binding domain.
Cullen
Bryan R
BR
HowardHughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
D015694:Gene Products, rev; D012322:RNA Precursors; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase