RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells

nature methods

Article

RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells

RNA localization and regulation are critical for cellular function, yet many live RNA imaging tools suffer from limited sensitivity due to background emissions from unbound probes. Here we introduce conditionally stable variants of MS2 and PP7 coat proteins (which we name dMCP and dPCP) designed to decrease background in live-cell RNA imaging. Using a protein engineering approach that combines circular permutation and degron masking, we generated dMCP and dPCP variants that rapidly degrade except when bound to cognate RNA ligands. These enhancements enabled the sensitive visualization of single mRNA molecules undergoing differential regulation within various subcompartments of live cells. We further demonstrate dual-color imaging with orthogonal MS2 and PP7 motifs, allowing simultaneous low-background visualization of distinct RNA species within the same cell. Overall, this work provides versatile, low-background probes for RNA imaging, which should have broad utility in the imaging and biotechnological utilization of MS2-containing and PP7-containing RNAs.

The regulation of mRNA abundance, localization and translation is crucial for many cellular behaviors. These include ensuring correct targeting of nascent proteins for secretion, controlling cell motility in response to external stimuli and facilitating synaptic plasticity between connected neurons. Knowing where and when these processes occur is critical for understanding the causes and effects of mRNA regulation. As such, methods that allow researchers to visualize the localization and dynamics of single mRNAs have become valuable tools for investigating RNA biology.

User-friendly imaging probes that can be readily implemented in living specimens are especially important for tracking mRNA dynamics in real time, with the most widely used tools being bacteriophage-derived components: the MS2 and PP7 coat proteins and their cognate MS2 and PP7 RNA hairpins. By expressing fluorescent protein-fused MCP or PCP, researchers can track single transcripts tagged with MS2 and/or PP7 arrays. An additional benefit of this strategy is that fluorescent proteins can be readily substituted with new sequences for multicolor imaging or effector functionality.

However, selectively visualizing tagged RNAs with these probes can be challenging due to background emissions from unbound coat proteins, which can hinder detection of RNA-bound species. To overcome this challenge, researchers have exploited nuclear localization signals to direct and sequester unbound coat proteins within the nucleus, allowing mature mRNAs to be visualized with increased contrast in the cytoplasm. Alternatively, RNA-specific contrast can be enhanced by increasing the number of fluorophores targeted to a given RNA. Although these strategies can enhance the detectability of tagged RNAs, they do not offer a solution to the broader challenge of mismatched coat protein-stem loop stoichiometries, a primary limiting factor in the specificity and utility of MS2-based and PP7-based technologies.

RNA-responsive reporter systems, such as fluorogenic aptamers and RNA-templated protein complementation, have been developed as alternatives to traditional coat protein-based strategies. In these approaches, signal generation is made RNA dependent, thus allowing users to track tagged RNAs and quantify their levels throughout cells. However, practical limitations make implementing these probes challenging in certain contexts. For example, aptamer-based systems require exogenous chromophores, which can be challenging to supply and maintain in certain cells and model systems. These methods can be further restricted due to the limited brightness and availability of their associated chromophores. Overall, an ideal system would combine the versatility of direct protein fusions with the reduced background and RNA-dependent signals of fluorogenic systems.

In more recent work, a labeling strategy that meets these criteria was developed based on so-called 'fluorogenic proteins'. In this approach, a conditionally stable RNA-binding protein is designed to be degraded unless bound to its target RNA, thereby reducing the levels of unbound proteins while rendering tagged RNAs visible via binding-induced reporter protein preservation. To develop such a probe, a virally derived Tat peptide was modified to contain a C-terminal degron, resulting in a nineteen-amino-acid sequence (called 'tDeg'), which renders proteins unstable while maintaining the ability to bind TAR-like RNA sequences (called 'Pepper'). Critically, binding to Pepper-tagged RNAs induces the selective stabilization of tDeg-tagged proteins, an effect that is facilitated via Pepper-mediated shielding of the degron, which, in turn, neutralizes its recognition by cellular machinery.

Recognizing the utility of multicolor RNA imaging and motivated by a growing need for improved RNA-binding tools, we set out to complement the tDeg strategy by developing destabilized versions of MCP and PCP. In natural contexts, MCP and PCP bind their cognate hairpins via orientations in which their N termini and C termini are solvent exposed. Thus, to render them conditionally stable, we implemented a two-step approach in which (one) circular permutation was used to reorient their termini to RNA-adjacent locations, followed by (two) the attachment and positional optimization of RNA-maskable C-degrons. Using this approach, we generated a conditionally stable MCP that is efficiently degraded by cells while undergoing a more than fifty-fold stability enhancement in response to MS2 binding. We confirmed the versatility of the domain by testing diverse fusions in fluorescence and bioluminescence assays. Furthermore, we exploited its RNA-dependent nature to sensitively visualize and record single mRNA dynamics throughout various subcellular locales, including within the nucleus and cytoplasm, and on the surfaces of mitochondria and the endoplasmic reticulum.

Finally, using a similar approach, we also generated a conditionally stable PCP. Equipped with two orthogonal and destabilized coat proteins, we exploited our new tools to simultaneously visualize distinct RNAs under low-background settings together in live cells. Overall, our approach combines the advantages of RNA-dependent signal generation, the versatility of genetic fusion and the well-characterized properties of RNA-binding coat proteins to enable sensitive and multicolor imaging of multiple distinct RNA species.