A key step in the evolution of complex organisms like eukaryotes was the organization of specific tasks into organelles. Reinkemeier et al. designed an artificial, membraneless organelle into mammalian cells to perform orthogonal translation. In response to a specific codon in a selected messenger RNA, ribosomes confined to this organelle were able to introduce chemical functionalities site-specifically, expanding the canonical set of amino acids. This approach opens possibilities in synthetic cell engineering and biomedical research.


Science, this issue p. eaaw2644


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Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes



The ability to engineer translation of noncanonical (unnatural) amino acids (ncAAs) site-specifically into proteins in living cells greatly expands the chemical space that can be used to control, tailor, and study cellular function. However, translation is a complex multistep process in which at least 20 different aminoacylated tRNAs, their cognate tRNA synthetases, ribosomes, and other factors need to act in concert to synthesize a polypeptide chain encoded by an mRNA transcript. To minimize interference with the host machinery, we aimed to engineer fully orthogonal translation into eukaryotes: to encode a new functionality in response to a specific codon in only one targeted mRNA, leading to site-specific ncAA incorporation only into the selected protein of choice. Although codon specificity can be achieved with genetic code expansion (GCE), this technology relies on using an orthogonal tRNA/tRNA synthetase pair (one that does not cross-react with any of the endogenous pairs) to reprogram a stop codon. Most commonly, the Amber (UAG) stop codon is used (20% abundance in human cells), and in principle, stop codon suppression can happen for every cytoplasmic mRNA that terminates naturally on this codon. Here, we present a strategy to generate a distinctly expanded genetic code for only selected mRNAs.



We hypothesized that it should be possible to create an orthogonal translation system by spatially enriching the key components of the GCE machinery in an orthogonally translating (OT) synthetic designer organelle and by targeting a specific mRNA to it. In order to perform protein translation, such an OT organelle would need to be readily accessible to the entire translational machinery of the host, thus precluding membrane encapsulation. Inspired by the concept of phase separation, which is used by cells to concentrate specific proteins and RNA locally, we hypothesized that it might be possible to use this principle to create such membraneless OT organelles. In our design, only a spatially distinct set of ribosomes associated with OT organelles can use the aminoacylated suppressor tRNA and thus will decode Amber codons only in the selected mRNA translated by the OT organelle, leading to a protein containing the ncAA.



To bring the modified suppressor tRNA and the translated mRNA of choice in close proximity to each other, we used different strategies to generate highly concentrated assemblies and spatial separation inside cells: (i) proteins undergoing phase separation in cells [fused-in sarcoma (FUS), Ewing sarcoma breakpoint region 1 (EWSR1), and spindle-defective protein 5 (SPD5), which contain long intrinsically disordered domains] and (ii) kinesin motor proteins, which spatially enrich at microtubule plus ends (KIF13A and KIF16B). We fused each of these to the suppressor tRNA synthetase as well as an RNA-binding domain major capsid protein (MCP) that binds to a specific RNA motif (ms2 loops) engineered into the untranslated region of the mRNA of choice, forming an ms2-MCP complex. Each of these approaches yielded the desired local enrichment and preferential stop codon suppression of the mRNA tagged with ms2 loops. However, by far the best performing system was a combination of phase and spatial separation, which typically formed a micrometer-sized organelle-like structure per cell. Cells that contained this organelle efficiently and selectively performed Amber suppression of only the targeted mRNA. We were able to demonstrate the utility and robustness of these OT organelles by selectively decoding any of the three stop codons in a variety of proteins with different ncAA functionalities in two different mammalian cell lines.



Our results show how to combine phase and spatial separation inside cells to allow the concentration of a custom designed task into a distinct specially designed membraneless organelle. We successfully demonstrated that specific and selective protein translation could be achieved within these OT organelles, which allowed the introduction of noncanonical functionalities into proteins in a codon-specific and mRNA-selective manner. The system only requires engineering five components into the cell and can be reprogrammed to other stop codons in a single step. We expect this concept to be a scalable platform for further organelle engineering and to provide a route toward generation of semisynthetic eukaryotic cells and organisms.


Membraneless OT organelles enable mRNA-specific GCE in eukaryotes.

OT organelles are designed organelles enriched in a suppressor tRNA/tRNA synthetase pair and a specific mRNA binding domain (MCP) by means of using an assembler protein (such as FUS and/or KIFs). A spatially distinct set of ribosomes associated with the OT organelle preferentially translates recruited mRNAs tagged with ms2 loops to yield the selected protein with the targeted site-specific noncanonical functionality.



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