Supplementary Figure 7

Real-time imaging of protein-RNA tethering. (a) Schematic representation of the protein-RNA tethered reporting system based on the interaction of tdMCP and the MS2 binding hairpin RNA. (b) Live cell imaging of Pepper-MS2. HeLa cells were transfected with plasmid expressing Pepper-MS2 and cultured for 30 hrs. Images were taken in the presence of 1 μM HBC620. Scale bar, 10 μm. (c) Imaging of HeLa cells expressing Pepper-MS2 and tdMCP-BFP targeted to different subcellular compartments: i, Histone 2B (Nucleic); ii, LifeAct (Filamentous actin); iii, α-tubulin; iv, a palmitoylation sequence (Membrane); v, a Golgi apparatus targeting signal; vi, TOMM20 (Outer mitochondrial membrane); vii, Srprb (ER membrane); viii, a peroxisomal targeting sequence. Scale bar, 10 μm. (d) Schematic representation of optogenetic manipulation of RNA localization. Upon blue light exposure, the CRY2-mCherry-tdMCP fusion protein interacts with plasma membrane-targeted CIBN and recruits Pepper-MS2 to the plasma membrane. Imaging (e) and quantification (f) of light-induced translocation of Pepper485 and mCherry to the plasma membrane. Both Pepper485 and mCherry fluorescence localized to the cytosol under dark conditions; however, they began accumulating at the plasma membrane upon light illumination. Cells were illuminated with a 458 nm laser (30 μW) spaced 17 min apart during three consecutive light-dark cycles. Scale bars, 10 μm. Data represent the mean ± s.d. (N = 5 cells). (g) Time course of CRY2-mCherry-tdMCP and Pepper-MS2 recruitment to the plasma membrane after illumination with a 458 nm laser (30 μW). Scale bar, 5 μm. (h) Quantification of plasma membrane and cytosolic CRY2-mCherry-tdMCP protein and Pepper-MS2 RNA in (g). Data represent the mean ± s.d. (N = 5 cells). (i) Fluorescence imaging of mCherry and Pepper485 in HeLa cells expressing CIBN, CRY2-mCherry-tdMCP, and Pepper-MS2 during dark recovery after initial blue light activation. Cells were firstly illuminated using a 458 nm laser (30 μW), and then imaged using a 900 nm two-photon excitation for Pepper485 fluorescence and a 561 nm excitation for mCherry fluorescence. Scale bar, 10 μm. (j) Quantification of plasma membrane and cytosolic CRY2-mCherry-tdMCP protein and Pepper-MS2 RNA. Data represent the mean ± s.d. (N = 5 cells). For b, c and i, at least two independent experiments were carried out with similar results.