Live-cell imaging of mammalian RNAs with Spinach2

Abstract

The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.

Keywords: Aptamer; Fluorescence microscopy; Live-cell imaging; RNA imaging; Spinach; Trinucleotide-repeat RNA.

Figure 1. Genetically encoded strategies for labeling RNA in living cells

A) The MS2-GFP imaging system involves labeling an mRNA of interest (black line) with up to 24 copies of the MS2 RNA hairpin in the 3′ UTR (gray line). When this tagged RNA is coexpressed with the MS2 coat protein fused to GFP, these hairpins are bound by MS2-GFP as dimers, leading to tagging by up to 48 MS2-GFP molecules. B) The PUM-HD split-FP imaging system involves engineering two PUM-HDs to distinct 8-nt regions in an RNA of interest. Each PUM-HD is then fused to half of a split fluorescent protein. When both PUM-HDs are bound to the target RNA, fluorescence complementation occurs and the RNA is labeled with a single fluorescent protein. C) The MS2/PP7 split FP imaging system involves tagging an RNA with both the MS2 RNA hairpin (gray hairpin) and the PP7 RNA hairpin (blue hairpin) in alternation. MS2 and PP7 coat proteins are each tagged with complementary halves of an FP. When both are bound, complementation occurs, leading to labeling of RNAs. D) Imaging an RNA of interest by Spinach tagging involves fusing Spinach to either the 5′ or 3′ end of an RNA and incubating cells with a dye that is nonfluorescent in solution. Only when the dye is bound to the tagged RNA does the Spinach-dye complex become fluorescent, specifically labeling the tagged RNA.

Figure 2. Schematic representations of the aptamers and dyes

A) The mFOLD-predicted secondary structures of Spinach (left) and Spinach2 (right) are shown. The green highlighted region shows the regions that contain mutations. B) DFHBI (left) and DFHBI-1T (right).

Figure 3. Spinach2-DFHBI-1T is brighter than Spinach2-DFHBI in live cells

A) Shown is a COS-7 cell expressing CGG₆₀-Spinach2. The cell was imaged using a widefield microscope with EGFP filter sets and a 100 msec exposure time. Cells were first incubated with 20 μM DFHBI and imaged. Media was then exchanged with media lacking dye for 30 min to remove DFHBI. This media was then supplemented with 20 μM DFHBI-1T for 30 min. Spinach2-DFHBI-1T images were collected following this incubation. B) Quantification of green fluorescence signal from Spinach2-DFHBI and Spinach2-DFHBI-1T in living cells. Scale bar, 20 μm. Images were used with permission from Song et al., 2014.

Figure 4. HEK 293T cells expressing 5S-Spinach2

Cells were incubated with 20 μM DFHBI for 30 min prior to imaging. Shown are green fluorescence (left) and differential interference contrast (DIC, right) images. Fluorescence image was collected by widefield microscopy with EGFP filter sets with a 1 sec exposure time. Scale bar, 10 μm.

Figure 5. CGG₆₀-Spinach2 colocalizes with mCherry-Sam68

Shown are images of COS-7 nuclei containing CGG₆₀ aggregates labeled with Spinach2-DFHBI or mCherry-Sam68. Spinach signal was collected by widefield microscopy with EGFP filter sets with a 100 msec exposure time. mCherry signal was collected using a Texas Red filter set and 200 msec exposure time. CGG₆₀-Spinach2 and mCherry-Sam68 are shown with and without DFHBI. Scale bar, 20 μm.

Figure 6. Examples of COS-7 nuclei with CGG₆₀-Spinach2 aggregates

Aggregates are highly heterogeneous and range in both size and number. Some representatives are shown. Images were collected by widefield microscopy with EGFP filter sets with 50–200 msec exposure times.