Open AccessA Cell-Based Method for Screening RNA-Protein Interactions: Identification of Constitutive Transport Element-Interacting Proteins
Author(s) -
Robert L. Nakamura,
Stephen G. Landt,
Emily Mai,
Jemiel Nejim,
Lily Chen,
Alan D. Frankel
Publication year - 2012
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0048194
Subject(s) - rna , cytoplasm , rna binding protein , microbiology and biotechnology , biology , reporter gene , chemistry , gene expression , gene , biochemistry
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.