Poster U18 - Synthetic biomolecular condensates enhance translation from a target mRNA in living cells

Introduction: 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 phase separation of intrinsically disordered proteins with a target mRNA that sequesters the mRNA into the condensate. This sequestration regulates gene expression by modulating translation of the sequestered mRNA or facilitating RNA processing mechanisms, such as splicing. Here, we engineer synthetic condensates using a fusion of an RNA-binding protein, the human Pumilio2 homology domain, and a synthetic intrinsically disordered protein, an elastin-like polypeptide, that exhibits liquid-liquid phase separation and can bind, and sequester, a target mRNA transcript.

Materials and

Methods: We characterize the Pum2-ELP condensate system using SPR, fluorescence microscopy, image analysis, microfluidics, biophysical assays (FRAP, FLIP, etc), biochemical assays (ELISA), and RNA-seq. We develop protocols for studying the effect of condensation on protein translation from a sequestered mRNA in water-in-oil cell-free protocells and inside E. coli cells. We use fluorescence microscopy to quantify reporter fluorescence and characterize the morphology of Pum2-ELP condensates. We use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in response to photobleaching (FLIP) experiments to characterize the material properties of the Pum2-ELP condensates. We develop a protocol for isolating and purifying synthetic condensates from E. coli using gradient ultracentrifugation and subsequently quantify the RNA in condensates and cell lysates using RNA-seq.

Results, Conclusions, and Discussions: We show that the Pum2-ELP fusion protein selectively binds an mRNA transcript of interest that contains multiple copies of a Pum2-binding RNA sequence at its 3’-end and sequesters the mRNA within a biomolecular condensate. Sequestration of the target mRNA within the condensate largely limits its translation in protocells. Conversely, and surprisingly, sequestration of the same target mRNA within Pum2-ELP biomolecular condensates increases its translation in E. coli. Characterization of synthetic condensates in E. coli show that synthetic Pum2-ELP ribonucleoprotein granules form through concentration-dependent liquid-liquid phase separation, are highly mobile, rapidly exchange biomolecules with the rest of the cell, preferentially sequester an mRNA of interest, and increase protein translation from the sequestered mRNA in E. coli cells. We present a mechanistic hypothesis for how our synthetic ribonucleotide granules increase protein translation by increasing ribosomal turnover within the condensate. This approach enables modulation of cell function via the formation of synthetic biomolecular condensates that can spatiotemporally regulate the expression of a target protein.

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