A Cell-Based Method for Screening RNA-Protein Interactions: Identification of Constitutive Transport Element-Interacting Proteins

<div><p>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. <em>In vitro</em> 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.</p> </div

CTE-interacting clones identified by the cDNA library screen.

<p>CTE-interacting clones identified by the cDNA library screen.</p

Analyses of non-coding library clones.

<p>Activities and specificities of the out-of-frame clones in the Tat-hybrid assay were determined as in Fig. 2. All fusions were independently tested using the HIV TAR CAT reporter to confirm that expression levels and transfection efficiencies were similar (data not shown).</p

<i>In vitro</i> binding of the CTE, hnRNP A1, and Tap.

<p>Gel shift experiments were performed with <i>in vitro</i> transcribed [³²P]-labelled CTE, GST-hnRNP A1, and/or GST-Tap in the binding reactions. Lanes 2–6 show a titration of GST-hnRNP A1 (0.045, 0.090, 0.18, 0.36, and 0.71 µM) and lanes 8–13 show a titration of GST-Tap (0.075, 0.15, 0.31, 0.63, 1.25, and 2.5 µM). In lanes 14–18, CTE complexes with 2.5 µM GST-Tap were titrated with hnRNP A1, resulting in a supershifted complex at the highest concentration (lane 18).</p

Properties of the out-of-frame library clones.

<p>Properties of the out-of-frame library clones.</p

<i>In vivo</i> binding of CTE-interacting clones.

<p>(Top) Activation assays with individual clones were performed on pHIV LTR CTE BTAR CAT (dark grey bars) and pHIV LTR BTAR CAT (light grey bars) reporters. Each clone was co-transfected along with the reporter DNA into HeLa cells and CAT activity was measured after 48 h. Plasmids encoding BIV Tat and Tap were included as positive controls. HIV Tat, a negative control, displayed a 2 to 3-fold preference for the CTE reporter, and thus library clones with specificities in that range were considered non-specific. To ensure that the expression of the library clones was consistent, each was independently co-transfected into HeLa cells with a pHIV LTR TAR-CAT reporter and CAT activity was found to be similar between all clones (data not shown). Library clones can activate transcription via HIV TAR because the fusions are to full-length Tat, which is able to bind HIV TAR. (Bottom) CTE specificity of each clone represents the ratio of activities on the CTE BTAR reporter to the BTAR reporter. Activities were normalized to 1 for BIV Tat specificity. The positive control Tat-Tap (1–372) fusion conferred the highest specificity for the CTE but other clones, including hnRNP A1, displayed similar levels of specificity.</p

Schematic diagram of the Tat-hybrid cDNA library screen.

<p>(A) Transcription elongation from the HIV LTR GFP reporter plasmid is enhanced only when a Tat-fusion protein interacts with the CTE RNA target, which is located in place of TAR at the 5′ end of the mRNA. The CTE reporter was integrated into HeLa cells to obtain a consistent, highly responsive cell line. The BIV TAR hairpin binds BIV Tat and serves as a positive control throughout the library screen and subsequent analyses of clones. (B) GFP activation level observed with a Tat-Tap positive control. SSC represents side scatter. (C) An HIV Tat-cDNA fusion library spiked with the Tap positive control (at 1∶100,000) was clonally introduced into the reporter cell line by protoplast fusion. CTE interacting clones were identified by FACS, plasmid DNA was isolated and re-introduced into <i>E. coli</i>, and a second round of library screening was carried out. Plasmids recovered after the second round of screening were identified as Tat-Tap clones by PCR. (D) cDNA library screen as in (C) except without the Tat-Tap positive control. Plasmids recovered after the second round sort were sequenced and further characterized.</p

Mapping the interaction domains of hnRNP A1.

<p>(A) Schematic diagram of the hnRNP A1 constructs (1–320, 1–194, 195–320, and 1–234) used for <i>in vitro</i> pull down or Tat-hybrid reporter experiments. (B) GST pull-down assays with hnRNP A1 and Tap. This experiment utilized GST-hnRNP A1 (1–320) or GST-Tap (1–619) and the corresponding [³⁵S]-labeled <i>in vitro</i> translated (IVT) proteins as indicated. The two input lanes (5 and 6) represent 1/10 of the <i>in vitro</i> translated proteins used for binding. GST alone was used as a negative binding control. (C) GST-Tap pull-down assays of full-length hnRNP A1 and two deletion mutants (1–194 and 1–234). The three input lanes (7–9) represent 1/10 of the <i>in vitro</i> translated proteins used for binding. GST alone was used as a negative binding control. (D) Activity and specificity of the hnRNP A1 truncations in the Tat-hybrid assay. Activities were determined using the same methods as in Fig. 2. Tap 1–372 and BIV Tat were included as positive controls and HIV Tat as a negative control.</p