Illuminating RNA Biology: Tools for Imaging RNA in Live Mammalian Cells

Abstract

The central dogma teaches us that DNA makes RNA, which in turn makes proteins, the main building blocks of the cell. But this over simplified linear transmission of information overlooks the vast majority of the genome produces RNAs that do not encode proteins and the myriad ways that RNA regulates cellular functions. Historically, one of the challenges in illuminating RNA biology has been the lack of tools for visualizing RNA in live cells. But clever approaches for exploiting RNA binding proteins, in vitro RNA evolution, and chemical biology have resulted in significant advances in RNA visualization tools in recent years. This review provides an overview of current tools for tagging RNA with fluorescent probes and tracking their dynamics, localization andfunction in live mammalian cells.

Keywords: RNA; chemical biology tools; genetically encoded tools; imaging; live-cell imaging; single-molecule tracking.

Figure 1:

Overview of tools covered in this review

Figure 2:

A&B) Genetically encoded tools can visualize tagged or untagged RNAs. C) With FPs, these systems use proteins to image RNA.

Figure 3:

A) Designed fluorophore-aptamer pairs leverage rigidification upon aptamer binding to product fluorescence turn-on. B) Fluorophores in these structures have a bond capable of cis-trans isomerization, where conformational flexibility renders the molecule nonfluorescent in bulk solution.

Figure 4:

A) Tethered fluorophore-quencher complexes utilize fluorophore-quencher pairs that primarily undergo contact quenching. This enables an aptamer binding event to reduce quenching and turn on fluorescence. B) Fluorophore-quencher pairs are constructed with modern fluorophores linked (usually via PEG) to a quenching moiety.

Figure 5:. Tracking mRNAs in live cells reveals nonuniform distributions in diverse cell types with implications for function.

(A) Two mRNAs were visualized in a live Drosophila oocyte with MS2/MCP and PP7/PCP. bcd mRNA (MS2/MCP-GFP, green) localizes to the anterior of the oocyte, whereas osk mRNA (PCP/PP7-mCherry, red) localizes to the posterior (arrow: autofluorescent yolk granules). Scale bar = 15 µm. Reprinted from (Abbaszadeh and Gavis, 2016) with permission from Elsevier. (B) Endogenous β-actin mRNA (24xMS2, MCP-TagRFPt, red) localizes to the leading edge in mouse embryonic fibroblasts. Free GFP (green) served as a cytoplasmic marker. Scale bar = 10 µm. Reprinted from (Katz et al., 2012), copyright Cold Spring Harbor Laboratory Press. (C) Movement of mRNA particles in dendrites of rat neurons is dynamic and indicative of targeted, bidirectional transport. The reporter 6xMS2-DsRed-3’UTR of Arc mRNA was cotransfected with MCP-GFP into rat cortical neurons to label mRNA (grey scale, upper panel). Intensely fluorescent spots correspond to gold beads used for biolistic transfection. Bottom panels: Kymographs of regions indicated by square and rectangular boxes on the top panel. Blue arrow heads: Moving mRNA particles (5 s intervals). Scale bar = 20 µm (top panel), 2 µm (inserts). Reprinted from (Dynes and Steward, 2008) with permission from Wiley. (D) Endogenous β-actin mRNA (24xMS2/tdMCP-GFP, arrow) is transferred between two mouse embryonic fibroblast cells via passage through nanotubes (right panel = zoom in of yellow box in the left panel). tdMCP-GFP was produced in the donor cell (bottom right), but not the acceptor cell (top left). Both cells are labeled with membrane targeted TagRFP-T (magenta). Scale bar = 5 µm. Reprinted from (Haimovich et al., 2017).

Figure 6:. Single mRNA tracking enables insights into mRNA life cycle processes.

(A) β-globin mRNA (24xPP7 in the intron, 24xMS2 in the 3’UTR) shows diffraction limited spots that are both green and red in the nucleus (left panel, arrow, scale bar = 4 µm). Fluorescence signal at the transcription site was recorded over time; fluctuations indicate stochastic transcription events. Reprinted from (Coulon et al., 2014). (B) Epithelial cells stably express the FLAG-SINAPS reporter to monitor protein translation at the single mRNA level. mRNA is labelled with 24xMS2 (red puncta). The encoded protein includes 24xSunTag at the N-terminus which binds scFV-GFP (green puncta represent single translation sites). Scale bar = 5 µm. From (Wu et al., 2016), reprinted with permission from AAAS. (C) NLS-MCP-Halo was produced to label mRNAs and ER-Turq2 is a marker for the ER. Gaussia mRNA (a protein trafficked through the secretory pathway) localizes to the ER, but only a small fraction of Renilla mRNA (a cytosolic protein) localizes to the ER. Scale bars = 5 µm. Reprinted from (Voigt et al., 2017) with permission from Elsevier. (D) Characterization of mRNA interaction dynamics with stress granules (SG) and P-bodies (PB). KDM5B-24xMS2 mRNA was labeled with NLS-MCP-Halo-JF646 (red). SGs and PBs were marked by fluorescent marker proteins (GFP-G3BP1, blue, and mRFP-DCP1a, green, respectively). Shown is a mRNA trajectory between two SGs and a PB over time (bottom panel). Three selected time points are indicated and cropped images are shown (top panel). Scale bar = 1 µm. Reprinted by permission from Springer Nature Customer Service Centre GmbH: (Moon et al., 2019). (E) The ‘TREAT siRNA reporter’ includes the Renilla coding sequence, 24xPP7 in the 3’UTR, followed by a siRNA site, and 24xMS2. Cells expressing the reporter also produce PCP-GFP (green) and MCP-Halo (magenta). Intact mRNA is dual colored (white) and was observed for several frames. Slicing of the RNA at the siRNA sites spatially separates PP7 and MS2 tags. Scale bar = 1 µm. Reprinted from (Horvathova et al., 2017) with permission from Elsevier.

Figure 7:. Live cell tracking of non-coding RNAs.

(A) The 3’end of Xist was tagged with 24xMS2 stem and stably integrated in mouse embryonic stem cells under control of a Doxycyclin-inducible promoter. Clusters of Xist RNA labeled with MCP-GFP 12 hours after induction are indicated by arrows (left panel). DNA is stained with Hoechst (red). Scale bar = 10 µm. Reproduced from (Ng et al., 2011). (B) The FASTmiR122 sensor consists of a modified Spinach RNA sequence that binds miR122, resulting in turn-on green fluorescence from the Spinach sensor in Huh7 cells (right panel). Reproduced from (Huang et al., 2017) by permission of Oxford University Press / RNA Society. (C) U1 snRNA was tagged at the 5’end with one copy of a minimal Riboglow RNA tag in HeLa cells and Cbl-5xPEG-ATTO 590 was added to live cells. Cytosolic U-bodies were induced by treatment with Thapsigargin. Scale bar = 5 µm. Reproduced from (Braselmann et al., 2018).