Abstract
This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA–protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA–protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA–protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens. The B3H assay builds on a well-established bacterial two-hybrid system for genetic analyses. As a result, protein–protein interactions can be assessed in tandem with RNA interactions with a bacterial two-hybrid assay to ensure that protein variants maintain their functionality. The B3H system is a powerful complement to traditional biochemical methods for dissecting RNA–protein interaction mechanisms: RNAs and proteins of interest do not need to be purified, and their interactions can be assessed under native conditions inside of a living bacterial cell. Once cloning has been completed, an assay can be completed in under a week and a screen in 1–2 weeks.
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References
Wagner, E. G. H. & Romby, P. Small RNAs in bacteria and archaea: who they are, what they do, and how they do it. Adv. Genet 90, 133–208 (2015).
Gottesman, S. & Storz, G. Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harb. Perspect. Biol. 3, a003798 (2011).
Ng Kwan Lim, E., Sasseville, C., Carrier, M.-C. & Massé, E. Keeping up with RNA-based regulation in bacteria: new roles for RNA binding proteins. Trends Genet. 37, 86–97 (2021).
Quendera, A. P. et al. RNA-binding proteins driving the regulatory activity of small non-coding RNAs in bacteria. Front. Mol. Biosci. 7, 78 (2020).
Olejniczak, M. & Storz, G. ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers new family of RNA matchmakers. Mol. Microbiol. 104, 905–915 (2017).
Holmqvist, E. & Vogel, J. RNA-binding proteins in bacteria. Nat. Rev. Microbiol. 16, 601–615 (2018).
Licatalosi, D. D., Ye, X. & Jankowsky, E. Approaches for measuring the dynamics of RNA–protein interactions. Wiley Interdiscip. Rev. RNA 11, e1565 (2020).
Ramanathan, M., Porter, D. F. & Khavari, P. A. Methods to study RNA–protein interactions. Nat. Methods 16, 225–234 (2019).
SenGupta, D. J. et al. A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl Acad. Sci. USA 93, 8496–8501 (1996).
Koh, Y. Y. & Wickens, M. Identifying proteins that bind a known RNA sequence using the yeast three-hybrid system. Methods Enzymol. 539, 195–214 (2014).
Hook, B., Bernstein, D., Zhang, B. & Wickens, M. RNA–protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening. RNA 11, 227–233 (2005).
Berry, K. E. & Hochschild, A. A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo. Nucleic Acids Res 46, e12 (2018).
Dove, S. L., Joung, J. K. & Hochschild, A. Activation of prokaryotic transcription through arbitrary protein–protein contacts. Nature 386, 627–630 (1997).
Dove, S. L. & Hochschild, A. Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa. J. Bacteriol. 183, 6413–6421 (2001).
Wang, C. D., Mansky, R., LeBlanc, H., Gravel, C. M. & Berry, K. E. Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA–DNA adapter protein. RNA 27, 513–526 (2021).
Pandey, S. et al. Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ. Nucleic Acids Res 48, 4507–4520 (2020).
Stein, E. M. et al. Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein. Nucleic Acids Res. 48, 7502–7519 (2020).
Faner, M. A. & Feig, A. L. Identifying and characterizing Hfq–RNA interactions. Methods 63, 144–159 (2013).
Kim, H. J., Chaulk, S., Arthur, D., Edwards, R. A. & Glover, J. N. M. Biochemical methods for the study of the FinO family of bacterial RNA chaperones. in RNA Chaperones (ed. Heise, T.) vol. 2106 1–18 (Springer US, 2020).
Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions. Nature 340, 245–246 (1989).
Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. PNAS 88, 9578–9582 (1991).
Mikulecky, P. J. et al. Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat. Struct. Mol. Biol. 11, 1206–1214 (2004).
Olejniczak, M. Despite similar binding to the Hfq protein regulatory RNAs widely differ in their competition performance. Biochemistry 50, 4427–4440 (2011).
Rao, X. et al. A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the β subunit and the primary σ subunit. Genes Dev. 23, 1818–1829 (2009).
Thibodeau, S. A., Fang, R. & Joung, J. K. High-throughput β-galactosidase assay for bacterial cell-based reporter systems. BioTechniques 36, 410–415 (2004).
Cadwell, R. C. & Joyce, G. F. Mutagenic PCR. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot4143 (2006).
Smirnov, A. et al. Grad-seq guides the discovery of ProQ as a major small RNA-binding protein. Proc. Natl Acad. Sci. USA 113, 11591–11596 (2016).
Holmqvist, E., Li, L., Bischler, T., Barquist, L. & Vogel, J. Global maps of ProQ binding in vivo reveal target recognition via RNA structure and stability control at mRNA 3′ ends. Mol. Cell 70, 971–982.e6 (2018).
Melamed, S., Adams, P. P., Zhang, A., Zhang, H. & Storz, G. RNA–RNA interactomes of ProQ and Hfq reveal overlapping and competing roles. Mol. Cell 77, 411–425.e7 (2020).
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).
Whipple, F. W. Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli. Nucleic Acids Res. 26, 3700–3706 (1998).
Acknowledgements
We thank members of the Berry lab for discussion and suggestions and A. Hochschild, P. Deighan and D. Heller for discussions and advice in establishing these protocols. This work was supported with funding from the National Institutes of Health [R15GM135878], the Camille and Henry Dreyfus Foundation, the Henry R. Luce Foundation and Mount Holyoke College.
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O.M.S., C.M.G. and K.E.B. organized, wrote and edited the manuscript. C.M.G. performed B3H experiments. O.M.S. and K.E.B. made figures.
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Nature Protocols thanks Simon Dove, Michelle Meyer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Berry, K. E. & Hochschild, A. Nucleic Acids Res. 46, e12 (2018): https://doi.org/10.1093/nar/gkx1086
Wang C. D. et al. RNA 27, 513–526 (2021): https://rnajournal.cshlp.org/content/27/4/513.full
Pandey S. et al. Nucleic Acids Res. 48, 4507–4520 (2020): https://doi.org/10.1093/nar/gkaa144
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Source Data Fig. 4c,d,e
Statistical source data (raw beta-galactosidase data).
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Stockert, O.M., Gravel, C.M. & Berry, K.E. A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA–protein interactions. Nat Protoc 17, 941–961 (2022). https://doi.org/10.1038/s41596-021-00657-4
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DOI: https://doi.org/10.1038/s41596-021-00657-4
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