title: A cell-based method for screening RNA-protein interactions: identification of constitutive transport element-interacting proteins.
abstract: We have developed a mammalian cell-based screening platform to identify proteins that assemble into RNA-protein complexes. Based on Tat-mediated activation of the HIV LTR, proteins that interact with an RNA target elicit expression of a GFP reporter and are captured by fluorescence activated cell sorting. This "Tat-hybrid" screening platform was used to identify proteins that interact with the Mason Pfizer monkey virus (MPMV) constitutive transport element (CTE), a structured RNA hairpin that mediates the transport of unspliced viral mRNAs from the nucleus to the cytoplasm. Several hnRNP-like proteins, including hnRNP A1, were identified and shown to interact with the CTE with selectivity in the reporter system comparable to Tap, a known CTE-binding protein. In vitro gel shift and pull-down assays showed that hnRNP A1 is able to form a complex with the CTE and Tap and that the RGG domain of hnRNP A1 mediates binding to Tap. These results suggest that hnRNP-like proteins may be part of larger export-competent RNA-protein complexes and that the RGG domains of these proteins play an important role in directing these binding events. The results also demonstrate the utility of the screening platform for identifying and characterizing new components of RNA-protein complexes.
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Nakamura
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Landt
authors.forename: Emily
authors.initials: E
authors.lastname: Mai
authors.forename: Jemiel
authors.initials: J
authors.lastname: Nejim
authors.forename: Lily
authors.initials: L
authors.lastname: Chen
authors.forename: Alan D
authors.initials: AD
authors.lastname: Frankel
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002469:Cell Separation; D002874:Chromosome Mapping / Q000379:methods; D003062:Codon; D003593:Cytoplasm / Q000378:metabolism; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000737:chemistry; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D003062:Codon; D018076:DNA, Complementary; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D012313:RNA; D005982:Glutathione Transferase