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Analyzing Mrna–Protein Complexes Using a Yeast Three-Hybrid System

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Analyzing Mrna–Protein Complexes Using a Yeast Three-Hybrid System Methods 26 (2002) 123–141 www.academicpress.com Analyzing mRNA–protein complexes using a yeast three-hybrid system David S. Bernstein, Natascha Buter, Craig Stumpf, and Marvin Wickens* Department of Biochemistry, University of Wisconsin—Madison, Madison, WI 53706, USA Accepted 22 January 2002 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. Ó 2002 Elsevier Science (USA). All rights reserved. 1. Introduction The three-hybrid system, like other genetic strategies, has the attractive feature that a clone encoding the The interactions of mRNAs and proteins are critical protein of interest is obtained directly in the screen. The for a wide variety of biological processes, ranging from system offers the possibility of connecting RNAs and developmental decisions to the proliferation of certain proteins on a broad and, perhaps, genomewide scale. viruses. For this reason, several methods have been de- The different genetic methods to detect RNA–protein veloped to analyze RNA–protein interactions using interactions have distinctive advantages. Several systems molecular genetics [1–10]. These complement an array of in eubacteria have been used to examine peptide–RNA biochemical approaches. interactions, and to select peptides with altered speci- We focus here on one such genetic method, the yeast ficities (e.g., [14,15]). To our knowledge, only the three- three-hybrid system [1]. In this method, an RNA–protein hybrid system has been used to identify new, naturally interaction in yeast results in transcription of a reporter occurring RNA binding proteins of biological signifi- gene. The interaction can be monitored by cell growth, cance. colony color, or the levels of a specific enzyme. Because In this article, we describe the system, summarize its the RNA–protein interaction of interest is analyzed in- published uses, and present protocols. We first present dependent of its normal biological function, a wide vari- principles and background, then discuss the key ele- ety of interactions are accessible. Published applications ments of the system including vectors, strains, and hy- of the method to date include: discovery of proteins that brid RNAs. Finally, we consider specific applications: bind to a known RNA sequence, confirmation of sus- analyzing a known interaction, finding a protein, finding pected interactions between an RNA and protein, muta- an RNA, and analyzing multiprotein complexes. The tional analysis of interacting RNAs and proteins, and utility of the system has benefited from adaptations in discovery and analysis of multiprotein:RNA complexes. many laboratories over the past few years. We relate In addition, three reports suggest that it should be feasible some of those developments, and hope to help others to identify the previously unknown RNA partner of an mold the system to new ends. RNA binding protein [11–13]. 2. Principles of the method * Corresponding author. Fax: 608-262-9108. The general strategy of the three-hybrid system is E-mail address: [email protected] (M. Wickens). diagrammed in Fig. 1A. DNA binding sites are placed 1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S1046-2023(02)00015-4 124 D.S. Bernstein et al. / Methods 26 (2002) 123–141 Fig. 1. Three-hybrid system to detect and analyze RNA–protein interactions. (A) General strategy of the system, (B) Specific protein and RNA components that typically have been used to date. Other RNA and protein components also can be used (see text). For simplicity, the following features are not indicated. Both lacZ and HIS3 are present in strain L40coat and YBZ-1 under the control of lexA operators, and so can be used as reporters. Multiple LexA operators are present (four in the HIS3 promoters and eight in the lacZ promoter), and LexA protein binds as a dimer. The hybrid RNA contains two MS2 binding sites, and MS2 coat protein binds as a dimer to a single site. Adapted, with permission from SenGupta et al. [1]. upstream of a reporter gene in the yeast chromosome. A teriophage MS2 coat protein, a small polypeptide that first hybrid protein consists of a DNA binding domain binds as a dimer to a short stem–loop sequence. The linked to an RNA binding domain. The RNA binding hybrid RNA (depicted in more detail in Fig. 3) consists domain interacts with its RNA binding site in a bi- of two MS2 coat protein binding sites linked to the functional (‘‘hybrid’’) RNA molecule. The other part of RNA sequence of interest, X. Hybrid protein 2 consists the RNA molecule interacts with a second hybrid pro- of the transcription activation domain of the yeast Gal4 tein consisting of another RNA binding domain linked transcription factor linked to an RNA binding protein, to a transcription activation domain. When this tripar- Y. tite complex forms at a promoter, even transiently, the Although the components depicted in Fig. 1B are reporter gene is turned on. Reporter expression can be most commonly used, others can be substituted. For detected by phenotype or simple biochemical assays. example, the MS2 components can be replaced by either The specific molecules most commonly used for the histone mRNA 30 stem–loop and the protein to three-hybrid analysis are depicted in Fig. 1B. The DNA which it binds (SLBP) (B. Zhang and M.W., unpub- binding site consists of a 17-nucleotide recognition site lished); the NRE in hunchback mRNA’s 30 untranslated for the Escherichia coli LexA protein, and is present in region (UTR) and its partner, Pumilio [16]; or hY5 and multiple copies upstream of both the HIS3 and LacZ its partner, Ro60 [17]. Similarly, the Gal4 DNA binding genes. Hybrid protein 1 consists of LexA fused to bac- domain can be used in place of LexA (B. Zhang and D.S. Bernstein et al. / Methods 26 (2002) 123–141 125 M.W., unpublished). We are not aware of a systematic analyzed, both in length and in specific sequence (see comparison of alternative components. Hybrid RNAs: Limitations): ideally, RNAs should be The three-hybrid approach has many of the same 150 or fewer nucleotides long, and should lack oligou- strengths and limitations of the two-hybrid system to ridylate stretches of 4 or more nucleotides. However, detect protein–protein interactions. By introducing li- these limitations are not universal: interactions have braries of RNA or protein, cognate partners can be been detected using RNAs up to 1600 nt in length [19]. identified. As in two-hybrid screens, the challenge then An alternative system exploiting an RNA polymerase II becomes identifying those that are biologically relevant. promoter has been described and should circumvent the For this purpose, mutations in the known RNA or sequence limitation [2]. protein component are very useful. Among the limita- tions of the system are certain technical constraints on 3.3. Screens producing hybrid RNAs and the low signal-to-noise ratio in screening a library when the genuine RNA– In screening cDNA or RNA libraries for interacting protein interaction is weak. partners, secondary screens of the initial ‘‘positives’’ are needed to winnow down candidates for further analysis (see Finding a Protein Partner for a Known RNA se- 3. Initial considerations quence, Step 5). One crucial secondary screen identifies positives requiring the ‘‘bait’’ to activate the reporter The three-hybrid system has been used to analyze and gene. Another screen identifies positives exhibiting the identify specific RNA-binding proteins for a number of correct sequence specificity. Subtle mutations that per- RNA targets. Table 1 summarizes directed tests of RNA turb the biological function of the bait are ideal. Subtly and protein partners, and Table 2 summarizes successful mutant baits will bind most biologically irrelevant, screens of cDNA libraries. Several general properties of ‘‘nonspecific’’ interactors, which can then be discarded. If the system merit a brief discussion at the outset; some subtle mutations are not available, cruder specificity tests are considered in greater detail later. (e.g., the antisense sequence) can be useful, almost sur- prisingly so (Table 2). Indeed, in the cDNA library 3.1. Strength of interactions screens carried out to date, virtually all positives that re- quired the RNA to activate and possessed the correct The affinities of RNA–protein interactions detected sequence specificity proved to be the genuine interactor by the three-hybrid system have Kd values from sub- (Table 2). This may mean that few proteins bind non- nanomolar to micromolar (Table 1). Although levels of specifically with sufficient affinity to register in the system. expression of the reporter gene generally correlate with measured in vitro affinities, the precise relationship be- tween them has not yet been reported. Reporter ex- 4. Key components: RNAs, vectors, and strains pression levels presumably vary with the abundance of the two partners, the influence of endogenous yeast 4.1. Hybrid RNAs proteins and RNAs, and the reporter gene used. (See Ref. [18] for a discussion of related points in the two- 4.1.1. General considerations hybrid system.) The RNA–protein interaction used to tether the hy- brid RNA to the promoter must be specific and of high 3.2. The hybrid RNA affinity. The RNA need not be structured.
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