Analyzing mRNA–protein complexes using a yeast three-hybrid system

doi:10.1016/S1046-2023(02)00015-4

Methods

Volume 26, Issue 2, February 2002, Pages 123-141

https://doi.org/10.1016/S1046-2023(02)00015-4 Get rights and content

Abstract

RNA–protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA–protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA–protein interaction, to analyze RNA–protein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.

Introduction

The interactions of mRNAs and proteins are critical for a wide variety of biological processes, ranging from developmental decisions to the proliferation of certain viruses. For this reason, several methods have been developed to analyze RNA–protein interactions using molecular genetics [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. These complement an array of biochemical approaches.

We focus here on one such genetic method, the yeast three-hybrid system [1]. In this method, an RNA–protein interaction in yeast results in transcription of a reporter gene. The interaction can be monitored by cell growth, colony color, or the levels of a specific enzyme. Because the RNA–protein interaction of interest is analyzed independent of its normal biological function, a wide variety of interactions are accessible. Published applications of the method to date include: discovery of proteins that bind to a known RNA sequence, confirmation of suspected interactions between an RNA and protein, mutational analysis of interacting RNAs and proteins, and discovery and analysis of multiprotein:RNA complexes. In addition, three reports suggest that it should be feasible to identify the previously unknown RNA partner of an RNA binding protein [11], [12], [13].

The three-hybrid system, like other genetic strategies, has the attractive feature that a clone encoding the protein of interest is obtained directly in the screen. The system offers the possibility of connecting RNAs and proteins on a broad and, perhaps, genomewide scale.

The different genetic methods to detect RNA–protein interactions have distinctive advantages. Several systems in eubacteria have been used to examine peptide–RNA interactions, and to select peptides with altered specificities (e.g., [14], [15]). To our knowledge, only the three-hybrid system has been used to identify new, naturally occurring RNA binding proteins of biological significance.

In this article, we describe the system, summarize its published uses, and present protocols. We first present principles and background, then discuss the key elements of the system including vectors, strains, and hybrid RNAs. Finally, we consider specific applications: analyzing a known interaction, finding a protein, finding an RNA, and analyzing multiprotein complexes. The utility of the system has benefited from adaptations in many laboratories over the past few years. We relate some of those developments, and hope to help others mold the system to new ends.

Section snippets

Principles of the method

The general strategy of the three-hybrid system is diagrammed in Fig. 1A. DNA binding sites are placed upstream of a reporter gene in the yeast chromosome. A first hybrid protein consists of a DNA binding domain linked to an RNA binding domain. The RNA binding domain interacts with its RNA binding site in a bifunctional (“hybrid”) RNA molecule. The other part of the RNA molecule interacts with a second hybrid protein consisting of another RNA binding domain linked to a transcription activation

Initial considerations

The three-hybrid system has been used to analyze and identify specific RNA-binding proteins for a number of RNA targets. Table 1 summarizes directed tests of RNA and protein partners, and Table 2 summarizes successful screens of cDNA libraries. Several general properties of the system merit a brief discussion at the outset; some are considered in greater detail later.

General considerations

The RNA–protein interaction used to tether the hybrid RNA to the promoter must be specific and of high affinity. The RNA need not be structured. Several different interactions have been used; of these, by far the most common has been that between a short stem–loop RNA structure and bacteriophage MS2 coat protein.

Three features of the MS2 coat protein and its cognate binding sites make this pair attractive.

High affinity and selectivity. The stem–loop binding site used in the three-hybrid system

Identifying and analyzing known interactors

Assaying the interaction between a specific RNA and protein is straightforward. A yeast strain with an integrated reporter gene and LexA/MS2 coat protein is transformed with two plasmids: an activation domain plasmid carrying the protein of interest, and a hybrid RNA carrying the desired RNA sequence. If the protein and RNA interact, the reporter is expressed. HIS3 and LacZ genes are the most common reporters used. Control experiments can be used to demonstrate that each component of the system

Finding protein partners of a known RNA sequence

The three-hybrid system can be used to identify a protein partner of a known RNA sequence. Typically, a three-hybrid reporter strain is created that expresses the RNA of interest as a hybrid molecule. A cDNA library is introduced into this strain by conventional transformation methods. When the RNA interacts with the protein produced from a cDNA, the HIS3 gene becomes active and the yeast grow on media lacking histidine and/or containing 3-AT. Additional selections can be performed using the

Finding an RNA partner for a known RNA binding protein

The three-hybrid system can be used to identify a natural RNA ligand for a known RNA-binding protein by screening an RNA library with a protein-activation domain fusion as bait. Although only three experiments of this type have been reported to date, the objective is sufficiently important that we discuss both the published work and our own experience. These experiments have obvious parallels to conventional in vitro reiterative selection methods, including SELEX and “genomic SELEX” [38], [39].

Multicomponent complexes

Multiple proteins often assemble on a single RNA to mediate the RNP's biological functions. Two proteins, P1 and P2, can interact with a single RNA in three permutations (Fig. 7). Each can be analyzed in the three-hybrid system.

Prospects

The simple three-hybrid system (Fig. 1) has been used to analyze a wide variety of known or suspected interactions, and has been extended to analyze complexes containing two or more proteins. This likely presages its application to even more complex situations. For example, complexes containing multiple RNAs might readily be analyzed and even more “layers” of protein likely can be penetrated to yield new components.

Further enhancements of the method may facilitate applications in several areas.

Acknowledgements

We are grateful to the Media Laboratory of the Biochemistry Department of the University of Wisconsin for help with figures, and to Carol Pfeffer with preparing the manuscript. We appreciate the helpful comments and suggestions of members of the Wickens laboratory. The laboratory is supported by research grants from the National Institutes of Health.

References (66)

B.R. Cullen
Methods Enzymol.
(2000)
S.G. Landt et al.
Methods Enzymol.
(2000)
H. Peled-Zehavi et al.
Methods Enzymol.
(2000)
C. Jain et al.
Methods Enzymol.
(2000)
D.W. Celander et al.
Methods Enzymol.
(2000)
H. Kollmus et al.
Methods Enzymol.
(2000)
E. Paraskeva et al.
Methods Enzymol.
(2000)
Y. Kang et al.
Virology
(1999)
C. Jain et al.
Cell
(1996)
P.D. Good et al.
Gene
(1994)

P. Bouffard et al.

RNA

(2000)

S.B. Rho et al.

RNA

(2000)

P.T. Lowary et al.

Nucleic Acids Res.

(1987)

Cited by (116)

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For complete details on the use and execution of this protocol, please refer to Zhang et al.¹
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Analyzing mRNA–protein complexes using a yeast three-hybrid system

Abstract

Introduction

Section snippets

Principles of the method

Initial considerations

General considerations

Identifying and analyzing known interactors

Finding protein partners of a known RNA sequence

Finding an RNA partner for a known RNA binding protein

Multicomponent complexes

Prospects

Acknowledgements

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Cell

Dev. Biol.

J. Biol. Chem.

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J. Biol. Chem.

J. Biol. Chem.

Mol. Cell

J. Biol. Chem.

Mol. Biochem. Parasitol.

Mol. Biochem. Parasitol.

Mol. Cell

Proc. Natl. Acad. Sci. USA

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