Development of the ALFA-array system to detect nascent translation in mammalian cells. (A) (1) Schematic of the CRISPR/Cas9 targeted insertion strategy to obtain endogenous DYNC1H1 gene tagged with 12X ALFA-tag. (2) Schematic of the ALFA-array system developed in mammalian cells. Twelve ALFA-tag sequences were multimerized (blue amino acid sequence) and inserted at the 5′ of the gene of interest sequence (DYNC1H1, yellow). (3) The NB-ALFA is expressed under spleen focus‐forming virus (SFFV) promoter fused to sfGFP and Streptococcal protein G (GB1) domain. (B, left) Micrographs of HeLa cells expressing a DYNC1H1 allele endogenously tagged 12x_ALFA-array tag and NB-ALFA-sfGFP, and treated (right) or not (left) with puromycin. (Green) NB-ALFA-sfGFP signal; (blue) DAPI. Scale bar, 5 µm. (Arrows) NB-ALFA-sfGFP spots. (Right) Quantification of the number of NB-ALFA-sfGFP spots per cell, with and without puromycin treatment. Error bars, standard deviation (n = 40 cells). (C) Micrographs of HeLa cells expressing a DYNC1H1 allele endogenously tagged 12x_ALFA-array tag and NB-ALFA-sfGFP, and hybridized in situ with a set of probes against DYNC1H1 mRNAs. On the merged panel: NB-ALFA-sfGFP signal (green); smFISH signals (red); DAPI (blue). Scale bar, 5 µm. (Arrows) DYNC1H1 polysomes. (D, top) Schematic of the MCP fusion used to image RNA. (Bottom) Maximum projection intensity images of cells stably expressing MCP-tdStayGold (on the left) or MCP-eGFP (on the right). All images were taken under the same conditions on an OMX wide-field microscope. Six time points were selected (0 min, 30 min, 60 min, 120 min, 150 min, and 180 min), and they are displayed with the same dynamic range. Red arrows point at transcription sites, and green arrows point at individual RNA molecules. Scale bar, 10 μm. (E) Intensities of single RNA molecules over time. Graph shows intensities of RNA molecules (mean and s.d. error; five cells). Because of bleaching, the single molecule signal could no longer be detected and measured after 120 min in the case of MCP-eGFP. Note that MCP-tdStayGold and MCP-eGFP signals were acquired with the same time frame frequency (see Materials and Methods). (F) The graph shows the number of single RNA molecules detected over time using FISHquant (five cells).
