Identification of Dengue RNA Binding Proteins Using RNA Chromatography and Quantitative Mass Spectrometry
A major challenge in dengue virus (DENV) research has been to understand the interaction of the viral RNA with host cell proteins during infection. Until recently, there were no comprehensive studies identifying host RNA binding proteins that interact with DENV RNA (Ward et al. RNA Biol 8 (6):1173–1186, 2011). Here, we describe a method for identifying proteins that associate with DENV RNA using RNA chromatography and quantitative mass spectrometry. The method utilizes a tobramycin RNA aptamer incorporated into an RNA containing the dengue 5′ and 3′ untranslated regions (UTRs) in order to reversibly bind RNA to a tobramycin matrix. The RNA–tobramycin matrix is incubated with SILAC-labeled cell lysates, and bound proteins are eluted using an excess of tobramycin. The eluate is analyzed using quantitative mass spectrometry, which allows direct and quantitative comparison of proteins bound to DENV UTRs and a control RNA–tobramycin matrix. This technique has the advantage of allowing one to distinguish between specific and nonspecific binding proteins based on the ratio of protein preferentially bound to the DENV UTRs versus the control RNA. This methodology can also be used for validation of quantitative mass spectrometry results using conventional Western blotting for specific proteins. Furthermore, though it was specifically developed to identify DENV RNA binding proteins, the RNA chromatography method described here can be applied to a broad range of viral and cellular RNAs for identification of interacting proteins.
Key wordsRNA affinity chromatography Dengue virus RNA binding proteins Tobramycin RNA aptamer Quantitative mass spectrometry SILAC
The authors would like to acknowledge Walter Blackstock for guidance with mass spectrometry and experimental design and Sheena Wee and Siok Ghee Ler in Quantitative Proteomics Group IMCB for technical assistance in mass spectrometry.
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