Detection of hnRNP H binding to its RNA binding site by FRET. (A) ECFP-tagged hnRNP H (ECFP-hnH) and EYFP-tagged MS2 (MS2-EYFP) proteins were coexpressed with HcRed1 containing MS2 and hnRNP H binding sites within its 3′ UTR. Each protein tag was imaged and visualized as indicated in each panel. EYFP was photobleached in a specific region (circled), and FRET efficiency was measured as an increase of ECFP fluorescence after EYFP photodestruction. Bars, 5 μm. (B) Quantification of FRET between ECFP-hnRH and MS2-EYFP coexpressed with different RNA-binding targets. FRET between ECFP-p80 and EYFP-p80 served as a positive control and coexpression of target RNA containing only MS2 binding sites (pRed-M4x) as a negative control. Cells expressing RNA with both target sequences (pRed-M6x-H5′) and ECFP-hnH only were analyzed as an additional negative control. The average and SEM are shown, and the number of assayed cells is indicated above the bars.
