ReviewNature protocols2022
A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions.
Review in Nature protocols, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
What it found
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The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
9 citing papers in PubMed, 9 citations in OpenAlex.
- Decoding RNA-protein interactions using high-throughput methods.RNA biology · 2026Review
- Molecular genetic characterization of bacterial KH-domain proteins.bioRxiv : the preprint server for biology · 2026Article
- Article
- Improved constructs for bait RNA display in a bacterial three-hybrid assay.Scientific reports · 2025Article
- Improved constructs for bait RNA display in a bacterial three-hybrid assay.bioRxiv : the preprint server for biology · 2024Article
- Generation of single-cysteinemicroPublication biology · 2024Article
- Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofRNA (New York, N.Y.) · 2023Article
- Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofbioRxiv : the preprint server for biology · 2023Article
- A translational repression reporter assay for the analysis of RNA-binding protein consensus sites.RNA biology · 2023Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.