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

. 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. We show that Pum2-ELP selectively binds RNA transcripts tagged with a PRS at their 3’ end. The Pum2-ELP fusion sequesters PRS-tagged RNA in a condensate in vitro in protocells containing an in vitro transcription-translation (IVTT) mixture. Sequestration of PRS-tagged mRNA encoding mCherry or superfolder green fluorescent protein (sfGFP) into a biomolecular condensate in an IVTT-containing protocell specifically inhibits expression of these proteins even in the presence of other RNA. Building on these in vitro results, we demonstrate that the synthetic RNPG can modulate the expression of target genes in E. coli . We show that Pum2-ELP forms condensates in E. coli that sequester mCherry-PRS encoding mRNA and reduces translation of mCherry compared to cells expressing mCherry mRNA without a PRS. To demonstrate modulation of cell function by the synthetic RNPG, we show that the Pum2-ELP fusion can sequester mRNA with a PRS that encodes a cytotoxic protein, Protein E 50,51 , and rescue cell viability compared to cells that express protein E from mRNA without a PRS. Finally, we purify synthetic RNPGs and use RNA-seq to confirm that the synthetic RNPGs preferentially sequester a PRS-tagged RNA of interest. Together, these results demonstrate the creation of a synthetic RNPG that can modulate cell function using only a minimal number of biomolecular components whose properties can be precisely tuned.