https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=pubmed&id=39350178&retmode=xml&tool=Litmetric&email=readroberts32@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09 3935017820241003
1754-16111812024Sep30Journal of biological engineeringJ Biol EngProbing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli.52525210.1186/s13036-024-00448-xRNA 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.© 2024. The Author(s).DolcemascoloRoswithaRInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.RuizRaúlRInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.BaldantaSaraSInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.GoirizLucasLInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.Pure and Applied Mathematics University Research Institute (IUMPA), Polytechnic University of Valencia, Valencia, 46022, Spain.Heras-HernándezMaríaMInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.Montagud-MartínezRoserRInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain.RodrigoGuillermoGInstitute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, Paterna, 46980, Spain. guillermo.rodrigo@csic.es.engJournal Article20240930
EnglandJ Biol Eng1013066401754-1611Post-transcriptional regulationRNA recognition motifSynthetic biologySystems biologyThe authors declare no competing interests.20245202024915202410165620241016552024101012024930epublish39350178PMC1144389510.1186/s13036-024-00448-x10.1186/s13036-024-00448-xEnglish MA, Gayet RV, Collins JJ. Designing biological circuits: synthetic biology within the operon model and beyond. Annu Rev Biochem. 2021;90:221–44.33784178Weber W, Fussenegger M. Emerging biomedical applications of synthetic biology. Nat Rev Genet. 2012;13:21–35.PMC709740322124480Roell MS, Zurbriggen MD. The impact of synthetic biology for future agriculture and nutrition. Curr Opin Biotechnol. 2020;61:102–9.31812911Peralta-Yahya PP, Zhang F, Del Cardayre SB, Keasling JD. Microbial engineering for the production of advanced biofuels. Nature. 2012;488:320–8.22895337Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.PMC402900224860555Cao J, Arha M, Sudrik C, Mukherjee A, Wu X, Kane RS. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells. Nucleic Acids Res. 2015;43:4353–62.PMC441718425845589Katz N, Cohen R, Solomon O, Kaufmann B, Atar O, Yakhini Z, Goldberg S, Amit R. Synthetic 5’ UTRs can either up- or downregulate expression upon RNA-binding protein binding. Cell Syst. 2019;9:93–106.31129060Dolcemascolo R, Heras-Hernandez M, Goiriz L, Montagud-Martinez R, Requena-Menendez A, Ruiz R, Perez-Rafols A, Higuera-Rodriguez RA, Perez-Ropero G, Vranken WF, Martelli T, Kaiser W, Buijs J, Rodrigo G. Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria. eLife. 2024;12:RP91777.PMC1094259538363283Montagud-Martinez R, Marquez-Costa R, Rodrigo G. Programmable regulation of translation by harnessing the CRISPR-Cas13 system. Chem Commun. 2023;59:2616–9.36757178Colognori D, Trinidad M, Doudna JA. Precise transcript targeting by CRISPR-Csm complexes. Nat Biotechnol. 2023;41:1256–64.PMC1049741036690762Yu L, Marchisio MA. CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits. Nucleic Acids Res. 2023;51:1473–87.PMC994365636651298Shotwell CR, Cleary JD, Berglund JA. The potential of engineered eukaryotic RNA-binding proteins as molecular tools and therapeutics. Wiley Interdiscip Rev RNA. 2020;11:e1573.PMC717541031680457Maris C, Dominguez C, Allain FHT. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 2005;272:2118–31.15853797Imai T, Tokunaga A, Yoshida T, Hashimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H. The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA. Mol Cell Biol. 2001;21:3888–900.PMC8705211359897Clingman CC, Deveau LM, Hay SA, Genga RM, Shandilya SMD, Massi F, Ryder SP. Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite. eLife. 2014;3:e02848.PMC409478024935936Liu Q, Schumacher J, Wan X, Lou C, Wang B. Orthogonality and burdens of heterologous AND gate gene circuits in E. coli. ACS Synth Biol. 2018;7:553–64.PMC582065429240998Ottoz DS, Rudolf F, Stelling J. Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res. 2014;42:e130.PMC417615225034689Stanton BC, Nielsen AAK, Tamsir A, Clancy K, Peterson T, Voigt CA. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Biol. 2014;10:99–105.PMC416552724316737Holmqvist E, Vogel J. RNA-binding proteins in bacteria. Nat Rev Microbiol. 2018;16:601–15.29995832Hoffmann F, Rinas U. Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol. 2004;89:73–92.15217156Jaswal K, Shrivastava M, Chaba R. Revisiting long-chain fatty acid metabolism in Escherichia coli: integration with stress responses. Curr Genet. 2021;67:573–82.33740112Gene Ontology Consortium. The gene ontology resource: 20 years and still GOing strong. Nucleic Acids Res. 2019;47:D330–8.PMC632394530395331Tomoyasu T, Ogura T, Tatsuta T, Bukau B. Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli. Mol Microbiol. 1998;30:567–81.9822822Ingolia NT, Hussmann JA, Weissman JS. Ribosome profiling: global views of translation. Cold Spring Harb Perspect Biol. 2019;11:a032698.PMC649635030037969Karp PD, Paley S, Caspi R, Kothari A, Krummenacker M, Midford P, Moore LR, Subhraveti P, Gama-Castro S, Tierrafria V, Paloma L, Muñiz-Rascado L, Bonavides-Martinez C, Santos-Zavaleta A, Mackie A, Sun G, Ahn-Horst TA, Choi H, Covert MW, Collado-Vides J, Paulsen I. The EcoCyc database in 2023. EcoSal Plus. 2023;11:eesp-0002.Minuesa G, Albanese SK, Xie W, Kazansky Y, Worroll D, Chow A, et al. Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia. Nat Commun. 2019;10:2691.PMC658450031217428Chavali PL, Stojic L, Meredith LW, Joseph N, Nahorski MS, Sanford TJ, Sweeney TR, Krishna BA, Hosmillo M, Firth AE, Bayliss R, Marcelis CL, Lindsay S, Goodfellow I, Woods CG, Gergely F. Neurodevelopmental protein Musashi-1 interacts with the Zika genome and promotes viral replication. Science. 2017;357:83–8.PMC579858428572454Paulus M, Haslbeck M, Watzele M. RNA stem-loop enhanced expression of previously non-expressible genes. Nucleic Acids Res. 2004;32:e78.PMC41963015163763Sleight SC, Bartley BA, Lieviant JA, Sauro HM. Designing and engineering evolutionary robust genetic circuits. J Biol Eng. 2010;4:12.PMC299127821040586Montagud-Martinez R, Ventura J, Ballesteros-Garrido R, Rosado A, Rodrigo G. Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time. J Biol Eng. 2020;14:12.PMC709815432226483Vind J, Sørensen MA, Rasmussen MD, Pedersen S. Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes: expression from reporter genes does not always reflect functional mRNA levels. J Mol Biol. 1993;231:678–88.7685825Espah Borujeni A, Zhang J, Doosthosseini H, Nielsen AAK, Voigt CA. Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage. Nat Commun. 2020;11:5001.PMC753623033020480Nikolados EM, Weiße AY, Ceroni F, Oyarzún DA. Growth defects and loss-of-function in synthetic gene circuits. ACS Synth Biol. 2019;8:1231–40.31181895Zearfoss NR, Deveau LM, Clingman CC, Schmidt E, Johnson ES, Massi F, Ryder SP. A conserved three-nucleotide core motif defines Musashi RNA binding specificity. J Biol Chem. 2014;289:35530–41.PMC427123725368328Lv X, Hueso-Gil A, Bi X, Wu Y, Liu Y, Liu L, Ledesma-Amaro R. New synthetic biology tools for metabolic control. Curr Opin Biotechnol. 2022;76:102724.35489308Kharas MG, Lengner CJ. Stem cells, cancer, and MUSASHI in blood and guts. Trends Cancer. 2017;3:347–56.PMC554614228718412Qi LS, Arkin AP. A versatile framework for microbial engineering using synthetic non-coding RNAs. Nat Rev Microbiol. 2014;12:341–54.24736794Thompson MG, Sedaghatian N, Barajas JF, Wehrs M, Bailey CB, Kaplan N, Hillson NJ, Mukhopadhyay A, Keasling JD. Isolation and characterization of novel mutations in the pSC101 origin that increase copy number. Sci Rep. 2018;8:1590.PMC578550729371642Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.PMC430204925516281Chothani S, Adami E, Ouyang JF, Viswanathan S, Hubner N, Cook SA, Schafer S, Rackham OJL. deltaTE: detection of translationally regulated genes by integrative analysis of Ribo-seq and RNA-seq Data. Curr Protoc Mol Biol. 2019;129:e108.PMC928569931763789Yu G, Wang L, Han Y, He Q. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–7.PMC333937922455463Supek F, Bosnjak M, Skunca N, Smuc T. REVIGO summarizes and visualizes long lists of Gene Ontology terms. PLoS ONE. 2011;6:e21800.PMC313875221789182