a cell-based method for screening rna-protein interactions identification of constitutive transport element-interacting proteins基于单元的方法筛选rna蛋白质相互作用本构运输element-interacting蛋白质的识别.pdfVIP

A Cell-Based Method for Screening RNA-Protein Interactions: Identification of Constitutive Transport Element-Interacting Proteins 1 1 2 1 2 1 Robert L. Nakamura , Stephen G. Landt , Emily Mai , Jemiel Nejim , Lily Chen , Alan D. Frankel * 1 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Biology, San Francisco State University, San Francisco, California, United States of America 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 platf