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Engineering Spatial Orthogonality into Protein Translation Elise M. Van Fossen,† Riley M. Bednar,† and Ryan A. Mehl* Department of Biochemistry and Biophysics, Oregon State University, 2011 Ag Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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leading to spatial or temporal orthogonality (Figure 1A). These additional “dimensions” of orthogonality have not yet been demonstrated for GCE, either because of their incompatibility with translation (in the case of temporal orthogonality) or because of the challenges of assembling all necessary translational components into a spatially exclusive location at sufficient concentrations to maintain orthogonal translation. Despite these technical challenges, in a recent publication, the Lemke group has taken a large step toward a spatial orthogonality approach for genetic code expansion through the utilization of phase separation.2 Spatial orthogonality is not a new concept for nature, as evidenced by the evolution of compartmentalization in various organisms (i.e., membranebound organelles), and it represents a viable avenue for achieving “perfect” orthogonality for various incompatible cellular processes. Engineering translation in a membrane-bound organelle, similar to what is used in the mitochondria, is challenging to orchestrate because all of the translational components need to be made in said organelle or transported through the membrane, and then the produced protein needs to be transported back to the desired location. Luckily, nature has shown that “perfect” orthogonality is not necessary in all instances and that a “spectrum” of orthogonality may be evolutionarily advantageous by allowing for errors under times of stress.3 A compelling example of this is the growing appreciation that liquid−liquid phase separation is implicated in the regulation of diverse biological functions by spatially segregating key components and processes without the sharply defined boundaries of traditional compartmentalized organelles.4 In these instances, molecular components can freely diffuse through the separated phase and may adopt different functions depending on their local concentration in the cytosol or in the separated phase.4 Inspired by these natural segregation processes and cognizant of the challenges associated with organizing GCE in membrane-bound organelles, Lemke’s group surmised that molecular orthogonality complications presented by introducing genetic code expansion into more complicated systems, such as eukaryotic cells, could be circumvented by sequestering key translational components of genetic code expansion [i.e., tRNA, aminoacyl tRNA-synthetase (aaRS), and mRNA] into membraneless, orthogonal translating (OT) organelles through a combination of phase separation and spatial enrichment strategies. To accomplish this, the authors chose two cellular, membrane-free compartmentalization strategies: one capitaliz-
dward Lemke and his team have recently reported the creation of a membraneless organelle for site-specific protein engineering, achieving a functioning level of spatial orthogonality for engineering translation. Establishing orthogonality, the mutual exclusivity of interactions between biomolecules within a living system, is key to the evolution and maintenance of complex processes in organisms. As synthetic biologists attempt to introduce engineered processes into living systems, they are also deeply invested in establishing orthogonal strategies to limit undesirable cross reactions. While most orthogonal approaches tend to focus on maintaining orthogonality by modulating molecular interactions between biomolecules, Lemke’s group developed a spatial orthogonality approach by using phase separation and spatial targeting to create a membraneless organelle for translation. The versatility of the membraneless organelle was highlighted by the production of three different proteins in two different eukaryotic cell lines, including a membrane protein. The development of a phase-separated, orthogonal organelle adds a new dimension of orthogonality to genetic code expansion, offers a new platform by which to study the process of translation, and outlines a new approach to orthogonalize additional cellular processes. A major goal of genetic code expansion (GCE, where the central dogma is re-engineered for the incorporation of nonstandard building blocks) is a fully orthogonal translational system wherein perfect, mutual exclusivity is maintained in the interactions between the engineered components and their endogenous analogues.1 Currently, attempts to achieve this level of orthogonality are mediated solely through a molecular orthogonality approach, in which undesirable interactions between engineered and endogenous components are minimized by meticulously tuning each engineered component to minimize off-target interactions with other translational components and improve on-target interactions (Figure 1A). Two main shortcomings of this approach limit its broad application in GCE and synthetic biology. First, all of the orthogonal and host components can interact freely in a shared cytosolic space, leading to unintended and often adverse interactions that are difficult to select against or decipher. Second, molecular orthogonality requires that the optimized individual components in one cellular system (e.g., in prokaryotes) transfer their optimized molecular orthogonality properties to other cell types (e.g., in eukaryotes) to maintain the uniquely designed and desired output; otherwise, new components must be made Molecular interactions are ultimately driven by both the intrinsic strength of the interactions and the cellular concentrations of the engineered and host components. Therefore, orthogonality can also be modulated by separating the engineered and host components in either space or time, © XXXX American Chemical Society
Received: July 3, 2019
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DOI: 10.1021/acs.biochem.9b00569 Biochemistry XXXX, XXX, XXX−XXX
Viewpoint
Biochemistry
Figure 1. The interplay of various forms of orthogonality available for genetic code expansion. (A) The three ‘dimensions’ of orthogonality. (B) The inverse requirements of molecular and spatial orthogonality along the molecular−spatial orthogonality plane. Several examples of varying degrees of spatial and molecular orthogonality are depicted along this spectrum. Solid arrows represent favorable interactions, hashed arrows represent unfavorable interactions, while blunted arrows indicate disallowed interactions. Increasing arrow width indicates increasing concentration.
ing on the phase separation properties of intrinsically disordered proteins and the other on the spatial enrichment of kinesin motor proteins. Selected targeting domains from these two classes of proteins (termed “assemblers”) were fused to both engineered aaRSs and to mRNA binding proteins that recruit a specific mRNA motif included on the mRNA of the gene of interest. As the assemblers naturally form an OT organelle, the engineered mRNA and aaRS would be spatially enriched while not being excluded from necessary translational machinery. The success of this strategy was determined in two ways: (1) by monitoring the production of reporter proteins created with GCE and (2) by monitoring the localization of GCE translational components to the OT organelle. Both membrane-free compartmentalization strategies showed marginal success in producing the reporter, but interestingly, a combination of both the phase separation and spatial organization strategies proved to be the most successful, yielding a micrometer-sized OT organelle and a significant improvement in reporter yield. Highlighting the versatility of the technology and minimal impact on protein production, this OT organelle was capable of producing three different proteins, including a membrane protein, in two different eukaryotic cell lines. More remarkably, by observing each component of translation independently, the authors were able to observe that even translational components, like orthogonal tRNA, independently localized to the OT organelle resulting in their reduced participation in native cytoplasmic translation. Moreover, Lemke’s group also realized that physically sequestering synthetic components may preserve the endogenous environment of the cell, decreasing the potential for experimental artifacts when monitoring cellular processes with GCE tools. To this end though, the consequences of creating and sustaining a micrometer-sized OT organelle may outweigh the benefits of decreasing the synthetic biology footprint of GCE through disruption of other cellular processes and/or organizations. The authors do note that as natural cellular phase separation strategies are better understood, the OT organelle can be designed to have a minimal impact on the cell. The Lemke group’s approach to engineer orthogonal translation in membraneless organelles is cunning because if spatial orthogonality works for translation, it provides the ability to obtain translational efficiency with lower molecular
orthogonality (Figure 1B), freeing GCE from the subset of aaRSs/tRNAs that possess molecular orthogonality in eukaryotic cells. This would add valuable flexibility to the selection of aaRSs and extend the toolset of noncanonical amino acids that can be incorporated. Moreover, the ability to compensate for low molecular orthogonality with enhanced spatial orthogonality may alleviate the stringent requirements for molecular orthogonality placed on current aaRSs, potentially opening access to more efficient but less fidelitous translational components. Future advancements can also be envisioned using this established framework to develop mechanisms to exclude certain components from the OT organelle. This could be used to increase the GCE translational efficiency by minimizing release factor-mediated translational termination or minimization of nonsense-mediated decay. More importantly, the Lemke group has demonstrated the feasibility and benefits of spatial orthogonality for the first time, inspiring future work to uncover additional means to engineer greater control over this form of orthogonality. The work also represents a conceptual advance for engineering spatial orthogonality into other processes. For example, one can imagine the use of phase separation-driven spatial sequestration of the components of the spliceosome (which had already been shown to form in a separated phase5) to engineer orthogonal splicing pathways, among many other applications. As synthetic biology moves forward, it is clear that controlling the orthogonality of complex cellular process will be an essential consideration. In our engineering of the central dogma, we should also recall that natural translation has not evolved to have ideal or perfect orthogonality, because this would remove a key mechanism to adapt under selective pressure. Rather, the natural translation has evolved to retain an acceptable level of orthogonality balanced by the needs of both efficient survival and adaptation. As we continue reengineering the central dogma, the stringency of orthogonality will be defined by the problem we are trying to solve. The work by Lemke and colleagues demonstrates multiple dimensions for controlling orthogonality are now not only accessible but also likely necessary for future engineering of more complex cellular processes. B
DOI: 10.1021/acs.biochem.9b00569 Biochemistry XXXX, XXX, XXX−XXX
Viewpoint
Biochemistry
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AUTHOR INFORMATION
Corresponding Author
*Department of Biochemistry and Biophysics, Oregon State University, 2011 Ag Life Sciences Building, Corvallis, OR 97331-7305. E-mail:
[email protected]. ORCID
Riley M. Bednar: 0000-0001-9452-825X Ryan A. Mehl: 0000-0003-2932-4941 Author Contributions †
E.M.V.F. and R.M.B. contributed equally to this work.
Funding
This work was supported by grants from National Institutes of Health Grant RGM114653A and National Science Foundation Grant MCB-1518265. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Liu, C. C., Jewett, M. C., Chin, J. W., and Voigt, C. A. (2018) Toward an orthogonal central dogma. Nat. Chem. Biol. 14, 103−106. (2) Reinkemeier, C. D., Girona, G. E., and Lemke, E. A. (2019) Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science 363, 6434−6443. (3) Gabaldon, T., and Pittis, A. A. (2015) Origin and evolution of metabolic sub-cellular compartmentalization in eukaryotes. Biochimie 119, 262−268. (4) Boeynaems, S., Alberti, S., Fawzi, N. L., Mittag, T., Polymenidou, M., Rousseau, F., Schymkowitz, J., Shorter, J., Wolozin, B., Van Den Bosch, L., Tompa, P., and Fuxreiter, M. (2018) Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol. 28, 420− 435. (5) Sawyer, I. A., Hager, G. L., and Dundr, M. (2017) Specific genomic cues regulate Cajal body assembly. RNA Biol. 14, 791−803.
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DOI: 10.1021/acs.biochem.9b00569 Biochemistry XXXX, XXX, XXX−XXX
