Fifteen years of the yeast three-hybrid system: RNA–protein interactions under investigation

Martin, Franck

doi:10.1016/j.ymeth.2012.07.016

Cited by 18 publications

(11 citation statements)

References 140 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The B3H assay established here will expand the tools currently available to investigate RNA–protein interactions ( 4 , 5 , 7 – 10 , 34 , 58 ) and complements previous techniques in ways that should extend their utility. As an alternative in vivo method to three-hybrid assays in yeast ( 1 , 2 , 8 , 22 ), our E. coli -based system offers high transformation efficiency as well as a distinct cellular machinery and environment to produce fusion proteins and hybrid RNAs. In particular, unlike yeast RNAP III, which is used to produce hybrid RNAs in the most commonly used yeast three-hybrid systems, E. coli RNAP can transcribe U-rich stretches and recognizes bacterial intrinsic terminators ( 27 ).…”

Section: Discussionmentioning

confidence: 99%

“…The establishment of a three-hybrid assay using bacterial cells offers potential to expand the range of RNA–protein interactions that can be investigated genetically. In particular, our B3H assay provides a complement to established yeast three-hybrid assays for studying RNA–protein interactions ( 1 , 2 , 8 , 22 ); as well as offering high transformation efficiency, the E. coli -based system enables the production of hybrid RNAs with bacterial intrinsic terminators at their 3′ ends, which cannot be produced in yeast. We anticipate that our bacterial system will facilitate the study of diverse RNA-binding proteins from bacteria and other organisms.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

Berry

Hochschild

2017

Nucleic Acids Research

View full text Add to dashboard Cite

The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA–protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein–protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA–protein interactions.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

Berry

Hochschild

2017

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…This powerful genetic method involves the expression in yeast cells of three chimerical molecules, which assemble to activate the reporter genes, HIS3 and LacZ (16,107,108). The first hybrid protein consists of a DNA-binding protein linked to an RNA-binding protein.…”

Section: Validation Of Ms Datamentioning

confidence: 99%

“…However, this approach requires a careful interpretation because the most common problem of Y3H analysis is the detection of a large number of false positive clones that may prohibit isolation of genuine RNA–protein partners. Therefore, it is necessary to perform additional control steps to confirm the biological relevance of the identified interaction (16,107,108). …”

Section: Validation Of Ms Datamentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

View full text Add to dashboard Cite

RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

show abstract

“…The development of programmable DNA binding domains such as TALEs (Transcription activator-like effectors) and dCas9 have revolutionized 1- and 2-hybrid approaches 8,13–15 . However, a challenge with n-hybrids is that each system needs to be carefully tuned and optimized for each new interaction, and more complex multicomponent systems, such as 3-hybrids, often lack in sensitivity and signal-to-noise 16,17 . Although 1-hybrids, and in special cases, 2-hybrids, can be utilized for synthetic biology purposes, in general, these methods are not suitable for applications where a high level of control and dynamic range is required.…”

mentioning

confidence: 99%

Evolution of a split RNA polymerase as a versatile biosensor platform

2017

View full text Add to dashboard Cite

Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

show abstract

Fifteen years of the yeast three-hybrid system: RNA–protein interactions under investigation

Cited by 18 publications

References 140 publications

A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Evolution of a split RNA polymerase as a versatile biosensor platform

Contact Info

Product

Resources

About