Synthetic ribonucleoprotein granules regulate translation of a target mRNA in living cells
Autor: | Seyed Eghtasadi, Daniel Shapiro, Sonal Deshpande, Junseon Min, David Fiflis, Dahlia Rohm, YIfan Dai, Aravind Asokan, Charles Gersbach, Ashutosh Chilkoti |
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Rok vydání: | 2022 |
DOI: | 10.21203/rs.3.rs-1982007/v1 |
Popis: | 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. |
Databáze: | OpenAIRE |
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