Yeast Three-hybrid System Research Articles

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Using the yeast three-hybrid system for the identification of small molecule-protein interactions with the example of ethinylestradiol.

Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo.

Hepatitis delta virus interacts with splicing factor SF3B155 and alters pre-mRNA splicing of cell cycle control genes.

Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs invivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.

Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5′ or 3′ untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5’-CAGWGH-3′ (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5′-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.

Alternative splicing regulation of doublesex gene by RNA-binding proteins in the silkworm Bombyx mori

ABSTRACTDoublesex is highly conserved and sex-specifically spliced in insect sex-determination pathways, and its alternative splicing (AS) is regulated by Transformer, an exonic splicing activator, in the model system of Drosophila melanogaster. However, due to the lack of a transformer gene, AS regulation of doublesex remains unclear in Lepidoptera, which contain the economically important silkworm Bombyx mori and thousands of agricultural pests. Here, we use yeast three-hybrid system to screen for RNA-binding proteins that recognize sex-specific exons 3 and 4 of silkworm doublesex (Bm-dsx); this approach identified BxRBP1/Lark binding to the exon 3, and BxRBP2/TBPH and BxRBP3/Aret binding to the exon 4. Investigation of tissues shows that BxRBP1 and BxRBP2 have no sex specificity, but BxRBP3 has – three of its four isoforms are expressed with a sex-bias. Using novel sex-specific silkworm cell lines, we find that BxRBP1 and BxRBP3 directly interact with each other, and cooperatively function as splicing repressors. Over-expression of BxRBP1 and BxRBP3 isoforms efficiently inhibits splicing of the exons 3 and 4 in the female-specific cells and generates the male-specific isoform of Bm-dsx. We also demonstrate that the sex-determination upstream gene Masc regulates alternatively transcribed BxRBP3 isoforms. Thus, we identify a new regulatory mechanism of doublesex AS in the silkworm, revealing an evolutionary divergence in insect sex-determination.

A modified yeast three-hybrid system enabling both positive and negative selections.

To increase the reporter repertoire of the yeast three-hybrid system and introduce the option of negative selection. Two new versions of the yeast three-hybrid systemwere made by modifying the MS2 coat RNA-binding protein and fusing it to the Gal4 DNA-binding protein. This allows the use of Gal4 inducible reporters to measure RNA-protein interactions. We introduced two mutations, V29I and N55K into the tandem MS2 dimer and an 11 amino acid deletion to increase RNA-protein affinity and inhibit capsid formation. Introduction of these constructs into the yeast strains MaV204K and PJ69-2A (which contain more reporters than the conventional yeast three-hybrid strains L40-coat and YBZ-1) allows RNA-protein binding interactions with a wide range of affinities to be detected using histidine auxotrophy, and negative selection with 5-fluoroorotic acid. This yeast three-hybrid system has advantages over previous versions as demonstrated by the increased dynamic range of detectable binding interactions using yeast survival assays and colony forming assays with multiple reporters using known RNA-protein interactions.

Identification of protein lysine methylation readers with a yeast three-hybrid approach

BackgroundProtein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete.ResultsThis study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei.ConclusionsOur approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers.

Introns provide a platform for intergenic regulatory feedback of RPL22 paralogs in yeast.

Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs.Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role.The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.

The Yeast Three-Hybrid System for Screening RNA-Binding Proteins in Plants.

Yeast-hybrid methods have been successfully applied for screening interacting partners of DNAs or proteins. A yeast-based method, the yeast three-hybrid system, using a chimeric protein of a DNA-binding domain (LexA or GAL4BD) with a protein (MS2 coat protein or HIV Rev. M10) having a hybrid RNA at the 3' end of a target RNA sequence, has been developed for screening RNA-binding proteins. When the target RNA interacts with RNA-binding proteins fused with an activation domain (AD), yeast cells having all the interacting components can survive on selection media, and interacting reporters, HIS3 and LacZ, are activated. Based on this selection, interaction can be easily monitored and detected by simple biochemical assays. The in vivo screening strategy has been widely applied for characterizing and evaluating specific interactions between target RNAs and RNA-binding proteins. Here, we describe a library screening strategy for isolating RNA-binding proteins of select target RNAs using the yeast three-hybrid method. We also describe strategies to verify binding specificity using both a yeast-dependent reporter system and a yeast-independent method, in vivo RNA immunoprecipitation (RIP).

The Yeast Three-Hybrid System for Protein Interactions.

Proteins rarely act alone as their functions tend to be regulated in vivo. Therefore, protein-protein interaction analyses provide key clues for understanding the complex biological processes in the living cell. Several techniques have been developed to elucidate the conformation of large protein complexes, dynamic protein complex rearrangement and transient protein interactions. Yeast two-hybrid system is a well-established method to analyze binary protein interactions. Here we describe a basic yeast three-hybrid method, which represents an additional refinement of the classical yeast two-hybrid system for analyzing further complex interactions among three proteins.

Chemical Dimerizers in Three-Hybrid Systems for Small Molecule-Target Protein Profiling.

The identification of the molecular targets and mechanisms underpinning the beneficial or detrimental effects of small-molecule leads and drugs constitutes a crucial aspect of current drug discovery. Over the last two decades, three-hybrid (3H) systems have progressively taken an important position in the armamentarium of small molecule-target protein profiling technologies. Yet, a prerequisite for successful 3H analysis is the availability of appropriate chemical inducers of dimerization. Herein, we present a comprehensive and critical overview of the chemical dimerizers specifically applied in both yeast and mammalian three-hybrid systems for small molecule-target protein profiling within the broader scope of target deconvolution and drug discovery. Furthermore, examples and alternative suggestions for typical components of chemical dimerizers for 3H systems are discussed. As illustrated, more tools have become available that increase the sensitivity and efficiency of 3H-based screening platforms. Hence, it is anticipated that the great potential of 3H systems will further materialize in important contributions to drug discovery.

The application of yeast hybrid systems in protein interaction analysis

Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein-protein, protein-DNA and protein-RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein-protein interactions, cytosol protein-protein interactions and membrane protein-protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years.

Yeast Three-Hybrid System for the Detection of Protein-Protein Interactions.

Protein-protein interaction studies provide useful insights into biological processes taking place within the living cell. A number of techniques are available to unravel large structural protein complexes, functional protein modules, and temporary protein associations occurring during signal transduction. The choice of method depends on the nature of the proteins and the interaction being studied. Here we present an optimized and simplified yeast three-hybrid method for analysis of protein interactions involving three components.

Identification of Psk2, Skp1, and Tub4 as trans-acting factors for uORF-containing ROK1 mRNA in Saccharomyces cerevisiae.

Rok1, a DEAD-box RNA helicase, is involved in rRNA processing and the control of cell cycle progression in Saccharomyces cerevisiae. Rok1 protein expression is cell cycle-regulated, declining at G1/S and increasing at G2. The downregulation of Rok1 expression in G1/S phase is mediated by the inhibitory action of two upstream open reading frames (uORFs) in the ROK1 5'-untranslated region (5'UTR). We identified Psk2 (PAS kinase), Skp1 (kinetochore protein) and Tub4 (γ-tubulin protein) as ROK1 5'UTR-interacting proteins using yeast three-hybrid system. A deletion analysis of PSK2 or inactivation of temperature-sensitive alleles of SKP1 and TUB4 revealed that Rok1 protein synthesis is repressed by Psk2 and Skp1. This repression appeared to be mediated through the ROK1 uORF1. In contrast, Tub4 plays a positive role in regulating Rok1 protein synthesis and likely after the uORF1-mediated inhibitory regulation. These results suggest that 5'UTR-interacting proteins, identified using three hybrid screening, are important for uORF-mediated regulation of Rok1 protein expression.

The Cytosolic Adaptor AP‐1A Is Essential for the Trafficking and Function of Niemann‐Pick Type C Proteins

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over-accumulation of low-density lipoprotein-derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three-hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP-1 via its acidic/di-leucine motif. Consequently, a nonfunctional AP-1A cytosolic complex resulted in a typical NPC-like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ-cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP-1A is essential for the lysosomal targeting and function of NPC1 and NPC2.

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription.

Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA

Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3′ untranslated region (UTR) of the RNA in mediating transport. Because the 3′ UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3′ UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3′ UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

Characterization of human septin interactions

Septins constitute a group of GTP binding proteins that assemble into homo- and hetero-oligomeric complexes and filaments. These higher order septin structures are thought to function like scaffolds and/or diffusion barriers serving as spatial localizers for many proteins with key roles in cell polarity and cell cycle progression. In this study, we extensively characterized septin interaction partners using yeast two-hybrid and three-hybrid systems in addition to precipitation analyses in platelets. As a result, we identified human hetero-trimeric septin complexes on a large scale, which had been only postulated in the past. In addition, we illustrated roles of SEPT9 that might contribute to hetero-trimeric septin complex formation. SEPT9 can substitute for septins of the SEPT2 group and partially for SEPT7. Mutagenic analyses revealed that mutation of a potential phosphorylation site in SEPT7 (Y318) regulates the interaction with other septins. We identified several septin-septin interactions in platelets suggesting a regulatory role of diverse septin complexes in platelet function.

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.