Fluorescence microscopy was performed on a Nikon A1R Laser Scanning Confocal Microscope with a 100x oil objective (1.45 NA, Plan Apo I), a pixel size of 0.25 μm and an integration time of 2.2 μsec unless otherwise noted below or in Supplementary Table 12 . Images were acquired at 16-bit depth with Nikon Elements Software and processed in ImageJ2 using the Fiji plugin. All live images were acquired with an environment chamber at 37°C. Laser lines used were 405 nm (DAPI, for nuclear staining and NLS-TagBFP imaging), 488 nm (GFP, for GFP-tagged proteins and Broccoli-tagged mRNA), 561 nm (TRITC, for ATTO 590 in Cbl-fluorophore probes and Alexa 546, Alexa 594 and Alexa 568 in FISH probes and secondary antibodies and for Halo-dyes JF595 and SiR594) and 638 nm (Cy5, for Cy5 and Halo-dye JF646). Image acquisition settings for each experiment are listed in Supplementary Table 12 .
For live imaging of Broccoli-tagged 5S and U6 RNA, published protocols were used21 (link). Briefly, plasmids pAVU6+27-F30-2xdBroccoli and pAV5S-F30-2xdBroccoli21 (link) were transfected in HEK 293T cells and split into imaging dishes 48 h post transfection. 24 h later, DFHBI-1T was added at a final concentration of 40 μM and cells were imaged under widefield illumination conditions as recommended21 (link) using a 60x oil objective and 250 ms exposure. No ND filters were used.
For live imaging of Broccoli-tagged 5S and U6 RNA, published protocols were used21 (link). Briefly, plasmids pAVU6+27-F30-2xdBroccoli and pAV5S-F30-2xdBroccoli21 (link) were transfected in HEK 293T cells and split into imaging dishes 48 h post transfection. 24 h later, DFHBI-1T was added at a final concentration of 40 μM and cells were imaged under widefield illumination conditions as recommended21 (link) using a 60x oil objective and 250 ms exposure. No ND filters were used.