Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells

Abstract

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the Cobalamin (Cbl) riboswitch as an RNA aptamer and a series of probes containing Cbl as a fluorescent quencher linked to a range of fluorophores. We demonstrate fluorescence turn-on upon RNA binding and proof of concept for tracking both mRNA and small U RNA in live cells. Main textThe complex spatiotemporal dynamics of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) affect virtually all aspects of cellular function. These RNAs associate with a large group of RNA binding proteins that dynamically modulate RNA localization, processing and function 1,2 . Such interactions govern mRNA processing, export from the nucleus, and assembly into translationally competent messages, as well as association into large macromolecular granules that are not translationally active, including P-bodies and stress granules (SGs) [3][4][5][6] . Similarly, uridine-rich small nuclear RNAs (U snRNAs, the RNA components of the spliceosome) 7 dynamically associate with protein components to comprise the functional spliceosomal complex in the nucleus 8 . During different stresses including bacterial infection, the U snRNAs along with the splicing machinery can be transiently sequestered in cytosolic foci All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/199240 doi: bioRxiv preprint first posted online Oct. 10, 2017; 3 called U-bodies 7 . Given the intricate connection between RNA localization, dynamics and function, there has been a strong push to develop tools for visualization of RNA in live cells to elucidate mechanisms underlying the mRNA and ncRNA life cycle.While there is a broad spectrum of tools to fluorescently tag proteins in live cells, far fewer approaches for live cell imaging of RNA exist. The most common system uses a series of tandem hairpins that bind an RNA-binding protein (MS2 or PP7 coat protein) coupled to a fluorescent protein (FP) [9][10][11] . This system has been used successfully to interrogate mRNA dynamics over time at the single molecule level 9,10 . However, one limitation of this system is that 12-24 copies of the aptamer are required to enhance fluorescence contrast, and the large size of the tag can perturb localization, dynamics and processing 12 of the RNA. An alternative approach involves attaching an aptamer to the RNA that directly binds a small molecular fluorogenic dye [13][14][15][16] . Recent years have seen the application of Spinach 17 , Broccoli 18 , and Mango 19 aptamers to label and track RNAs in live mammalian cells. However, all these aptamers evolved in vitro contain a G-quadruplex 20 , which has been shown to complicate RNA folding and ligand selectivity in mammalian cells [21][22...

Evaluating Riboglow-FLIM probes for RNA sensing

“…2). 14,16,17 In our proof-of-concept study for Riboglow-FLIM in live cells, we showed compatibility with RNA multiplexing by exploiting the phylogenetic diversity of Cbl riboswitches. 20 We recently expanded the system to demonstrate the potential of Riboglow-FLIM for RNA visualization in complex multicellular systems.…”

“…To complement existing tools, we developed Riboglow 14,15 and recently expanded the platform to visualize model RNAs in commonly investigated U-2 OS cells using fluorescence lifetime imaging microscopy (FLIM). 16,17 The components of Riboglow are a genetically encoded RNA tag and a small molecule consisting of the ligand cobalamin (Cbl) that is synthetically coupled to a fluorophore (here, ATTO 590 or Cy5).…”

“…16,17 The components of Riboglow are a genetically encoded RNA tag and a small molecule consisting of the ligand cobalamin (Cbl) that is synthetically coupled to a fluorophore (here, ATTO 590 or Cy5). 14 The RNA element in Riboglow is derived from a bacterial riboswitch, 18 and as a result, the RNA tag is short and takes advantage of the selective and high-affinity binding between the riboswitch and its cognate ligand Cbl. 14 The Cbl ligand is also a fluorescence quencher.…”

“…1 and 2B). 14,16,17 Binding of the RNA ligand to Cbl affects this fluorescence signal, such that the intensity and lifetime readouts upon RNA tag binding change (Fig. 2).…”

“…Riboglow is compatible with a range of synthetic fluorophores and linkers, where the fluorescence quencher Cbl can be fused to various fluorophores using different linkers. 14 Riboglow-ATTO 590 and Riboglow-Cy5 probes were designed previously and proof-of-concept studies revealed that RNAs that are genetically tagged with Riboglow can be sensed by an increase in fluorescence intensity or fluorescence lifetime in live mammalian cells. 17 We recently demonstrated that Riboglow-FLIM 16 offers the ability to quantitatively visualize RNAs in vitro and in live U-2 OS cells using the Cbl-4xGly-ATTO 590 probe, expanding the usability of the Riboglow platform to FLIM.…”