Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

Reinkemeier, Christopher D.; Lemke, Edward A.

doi:10.1016/j.cell.2021.08.001

Cited by 39 publications

(39 citation statements)

References 119 publications

(169 reference statements)

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“…However, one downside of this technique is that it is not mRNA-specific, leading to potential background labeling of untargeted proteins with their naturally occurring stop codons being suppressed. To tackle this problem, we utilized our recently developed synthetic orthogonally translating (OT) film-like organelles to form a distinct protein translational machinery on the outer mitochondrial membrane surface ( 36 ). These organelles exclusively reassigned two Amber codons for the target FG-NUP and incorporated TCO*A at the two specified sites with high selectivity, ensuring minimal interference for endogenous protein translation and negligible background staining (Fig.…”

Section: Resultsmentioning

confidence: 99%

Deciphering the conformations and dynamics of FG-nucleoporins in situ

Heidari

Mikhaleva

et al. 2022

Preprint

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The ~120 MDa nuclear pore complex (NPC) acts as a gatekeeper for the molecular traffic between the nucleus and the cytosol. Small cargo readily passes through the transport channel, yet large cargo requires specialized nuclear transport receptors. While the scaffold structure that anchors the NPC in the double-layered nuclear envelope has been resolved to remarkable details, the spatial organization of intrinsically disordered nucleoporins (NUPs) within the central channel remains enigmatic. These so-called FG-NUPs account for about one-third of the total mass of the NPC and form the actual transport barrier. Here we combined site-specific fluorescent labeling in non-fixed cells and fluorescent lifetime imaging microscopy (FLIM) to directly decipher the conformations of an essential constituent of the permeability barrier, NUP98, inside the functioning NPCs using Fluorescence resonance energy transfer (FRET). With detailed measurements of the distance distribution of eighteen NUP98 segments combined with coarse-grained modeling, we mapped the uncharted biochemical environment inside the nanosized transport channel. We found that "good-solvent" conditions for a polymer dominate the inside of the nanosized NPC, expand the FG-domain in situ and facilitate nuclear transport, in sharp contrast to the collapsed NUP98 FG-chain in aqueous solution. The combination of fluorescence microscopy, high-resolution electron tomography, and molecular simulation opens a window into the so-far unresolved organization of the FG-NUPs at the center of NPC function, allowing us to reconcile scientific models of nuclear transport.

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Section: Resultsmentioning

confidence: 99%

Deciphering the conformations and dynamics of FG-nucleoporins in situ

Heidari

Mikhaleva

et al. 2022

Preprint

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“…This study has several antecedents, though it differs from them in significant ways. An elegant set of studies by Lemke and coworkers showed that a synthetic RNPG 70,71 could successfully recruit a target mRNA to a protein condensate to spatially limit the incorporation of non-canonical amino acids in the protein encoded by the mRNA within the condensate, but they used a naturally occurring IDP with a limited range, and thus tunability, of phase behavior. Navarro et al used Pum1, a homolog of Pum2, to sequester endogenous mRNA into a synthetic condensate 28 .…”

Section: Discussionmentioning

confidence: 99%

Synthetic ribonucleoprotein granules regulate translation of a target mRNA in living cells

Eghtasadi

Shapiro²,

Deshpande

et al. 2022

Preprint

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Biomolecular condensates composed of proteins and RNA, known as ribonucleoprotein granules, are one approach by which cells regulate post-transcriptional gene expression. The formation of ribonucleoprotein granules typically involves the liquid-liquid phase separation of intrinsically disordered proteins with a target mRNA, sequestering the mRNA into the condensate. This sequestration regulates gene expression by inhibiting translation or facilitating RNA processing, such as splicing. Here, we designed a recombinant fusion of the human Pumilio2 homology domain (Pum2) RNA-binding protein and a synthetic intrinsically disordered protein that exhibits liquid-liquid phase separation to create synthetic ribonucleoprotein granules that extrinsically regulate the expression of a target gene. We show that this fusion protein selectively binds an RNA transcript of interest that contains a Pum2-binding RNA sequence at its 3’-end and sequesters the mRNA within a biomolecular condensate. Sequestration of a target mRNA within the condensate largely reduces the translation of the target RNA in protocells and in E. coli compared to cells that do not sequester the target mRNA in a synthetic condensate. We demonstrate that the viability of E. coli that harbor a cytotoxic protein can be rescued by sequestering the mRNA encoding the cytotoxic protein within a synthetic condensate. Finally, we use RNA-seq to determine that the target RNA of interest is preferentially sequestered in synthetic ribonucleoprotein granules. This approach enables modulation of cell function via the formation of a synthetic biomolecular condensate that spatiotemporally regulates the expression of a target protein.

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“…52−54 The potential for incorporation in response to endogenous amber codons can be partially prevented by the cellular compartmentalization of the mRNA using liquid-phase segregation or membrane recruitment. 55,56 However, the problem of cells incorporating natural amino acids at TAG codons is well known, particularly for Phe and Tyr analogues. Though background-causing amino acids may be dropped out of the media in E. coli, 11,57 methods for addressing this problem in eukaryotic species have not been reported.…”

Section: ■ Introductionmentioning

confidence: 99%

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

et al. 2022

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Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

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Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

Cited by 39 publications

References 119 publications

Deciphering the conformations and dynamics of FG-nucleoporins in situ

Deciphering the conformations and dynamics of FG-nucleoporins in situ

Synthetic ribonucleoprotein granules regulate translation of a target mRNA in living cells

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

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