TriFC analysis for studying RNA-protein interactions in live cells. (A) Depiction of the TriFC analysis employed in the study. The mRNA-reporter molecule contains HIV-1 vRNA packaging signal psi in close proximity to the integrated MS2 RNA-binding site (MS2BS). MS2-VN binds MS2BS to tether to the mRNA molecule. The C-terminal moiety of Venus (VC) is expressed as hGag-VC fusion and binding of hGag-VC to the RNA packaging sequence (psi domain) will bring both VN and VC Venus moieties in close enough proximity to generate a fluorescent signal by VN-VC complementation. (B) A 19-basepair deletion from SL3 of psi prevents the binding between hGag-VC and psi RNA and does not yield fluorescence complementation. (C) HeLa cells were grown on coverslips and transfected with pGL3MS2site-psi, MS2-VN and Gag-VN. 24 hr later laser scanning confocal microscopy was used to assess TriFC. Bright fluorescence signals in cytoplasmic punctae were detected indicating that the interaction between hGag-VC and the psi RNA domain occurred. This condition represented a positive control for the TriFC system employed here. TriFC signals were not detected when HeLa cells were transfected with the mutated psi reporter (pGL3MS2site-Delta-psi) (D). In order to determine the specificity of the TriFC assay, HeLa cells were co-transfected with plasmids expressing either Staufen1-VC (E) or IMP1-VC (F) along with MS2-VN and pGL3MS2site-psi. TriFC was absent in all cells examined indicating that Staufen1 and IMP1 do not bind the psi RNA. In parallel experiments, Gag did not associate with mRNA reporter bearing β-Actin zipcode sequence (pGL3-βActin zipcode) (G) whereas IMP1 did (H), as expected . Phase contrast images are shown on the left of each panel in (C)-(H). The size bars are equal to 10 μm.