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| Status |
Public on Oct 09, 2024 |
| Title |
Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli [RNA-seq] |
| Organism |
Escherichia coli |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli. Here, employing high-throughput RNA sequencing, we explored the impact of Musashi expression on the transcriptomic and translatomic profiles of E. coli, revealing certain metabolic interference, induction of post-transcriptional regulatory processes, and spurious protein-RNA interactions. Engineered Musashi protein mutants displayed compromised regulatory activity, emphasizing the importance of both RRMs for specific and sensitive RNA binding. We found that a mutation known to impede allosteric regulation led to similar translation control activity. Evolutionary experiments disclosed a loss of function of the synthetic circuit in about 40 generations, with the gene coding for the Musashi protein showing a stability comparable to other heterologous genes. Overall, this work expands our understanding of RRMs for post-transcriptional regulation in prokaryotes and highlight their potential for biotechnological and biomedical applications.
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| Overall design |
To investigate the effect of Musashi on Escherichia coli gene expression, a vector encoding (or devoid of) MSI gene was transformed into JS006 strain.
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| Contributor(s) |
Ruiz R, Dolcemascolo R, Baldanta S, Goiriz L, Heras-Hernández M, Montagud-Martínez R, Rodrigo G |
| Citation(s) |
39350178 |
| |
| Submission date |
Aug 23, 2024 |
| Last update date |
Oct 09, 2024 |
| Contact name |
Guillermo Rodrigo |
| E-mail(s) |
guillermo.rodrigo@csic.es
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| Organization name |
Agencia Estatal Consejo Superior de Investigaciones Científicas
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| Street address |
Calle del Catedrático Agustín Escardino Benlloch, 9
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| City |
Paterna |
| ZIP/Postal code |
46980 |
| Country |
Spain |
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| Platforms (1) |
| GPL25368 |
Illumina NovaSeq 6000 (Escherichia coli) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA1151706 |
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