Yeast Three-hybrid System Research Articles

Screening of a cytokinin binding protein using the yeast three-hybrid system

Screening of a cytokinin binding protein using the yeast three-hybrid system

Screening of a cytokinin binding protein using the yeast three-hybrid system

Screening of a cytokinin binding protein using the yeast three-hybrid system

19] Using the yeast three-hybrid system to detect and analyze RNA-protein interactions

The interactions of ribonucleic acids (RNAs) and proteins are critical for a wide variety of cellular processes ranging from translation to the proliferation of certain viruses. For this reason, several methods have been developed to facilitate the analysis of RNA–protein interactions by genetic means. These complement an array of biochemical approaches, including RNA affinity chromatography. This chapter discusses one such genetic method—namely, the yeast three-hybrid system. In this method, an RNA–protein interaction in yeast results in the 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. The published applications of the method to date include the discovery of proteins that bind to known RNA sequences, confirmation of suspected interactions between an RNA and protein, and mutational analysis of interacting RNAs and proteins. In addition, one report suggests that it may be feasible to identify the previously unknown RNA partner of an RNA-binding protein and to find protein components of multiprotein-RNA complexes in which both protein–protein and RNA–protein interactions are important.

Improvement of a yeast three-hybrid system to isolate an anxin receptor

Improvement of a yeast three-hybrid system to isolate an anxin receptor

8] Use of a yeast three-hybrid system to clone bridging proteins

The three-hybrid system described here should facilitate screening cDNA libraries to clone "bridging" proteins in ternary complex. The pDela constructs were initially designed to be compatible with the Gal4-based two-hybrid system, such as pGAD424/pGBT9. The Ura selection offered by pDela makes it easy to introduce the "bridging" protein into the yeast containing the other two hybrids, the BD fusion protein and the AD fusion protein. Because many of the widely used two-hybrid systems employ Trp-, Leu-, His-, or ziocin selection in the shuttle vectors, the Ura(+)-based pDela constructs can be used directly in those systems.

Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.

27] Yeast three-hybrid system to detect and analyze RNA-protein interactions

27] Yeast three-hybrid system to detect and analyze RNA-protein interactions

Improvement of a yeast three-hybrid system to isolate an auxin receptor

Improvement of a yeast three-hybrid system to isolate an auxin receptor

Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA).

We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.

The gag domains required for avian retroviral RNA encapsidation determined by using two independent assays.

The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity. Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation.

Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3′ splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

Regulation of eukaryotic protein synthesis: selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1.

To better understand regulation of eukaryotic protein synthesis, we studied cellular and viral mRNA translation in influenza virus-infected cells. Influenza virus infection results in a dramatic shut-off of cellular protein synthesis that is concomitant with selective viral mRNA translation. Earlier work showed that these events are mediated by viral and/or cellular factors binding to the 5' untranslated region (5' UTR) of viral mRNAs. To identify trans-acting cellular proteins responsible for selective viral protein synthesis, we employed the yeast three-hybrid system. Using the 5' UTR of the influenza virus nucleocapsid protein (NP) mRNA as bait, we identified the cellular RNA-recognition motif containing RNA-binding protein G-rich sequence factor 1 (GRSF-1) as a positive-acting translational regulatory factor. The in vivo yeast assay revealed GRSF-1 specifically bound to the NP 5' UTR but not select NP 5' UTR mutants or cellular RNA 5' UTRs. These data were confirmed by gel shift assays using recombinant GRSF-1. Importantly, recombinant GRSF-1 specifically stimulated translation of a NP 5' UTR-driven template in cell-free translation systems. Furthermore, translation efficiency of NP 5' UTR-driven templates was reduced markedly in GRSF-1-depleted HeLa cell extracts, but restored in GRSF-1-reconstituted extracts. GRSF-1 also stimulated translation of an NP 5' UTR-driven template in HeLa cell extracts that were depleted of essential factors by addition of RNA oligonucleotides representing the viral 5' UTR RNA. Taken together, these data document the functional demonstration of a cellular protein binding to influenza virus RNAs and, importantly, suggest that influenza virus may recruit GRSF-1 to the 5' UTR to ensure preferential translation of viral mRNAs in infected cells.

Identification of RNAs that bind to a specific protein using the yeast three-hybrid system.

We have adapted the yeast three-hybrid system to identify RNA ligands for an RNA-binding protein. In this assay system, a protein-RNA interaction is detected by the reconstitution of a transcriptional activator using two hybrid proteins and a hybrid RNA. The RNA molecule is tethered to the promoter of a reporter gene by binding to a hybrid protein consisting of the bacteriophage MS2 coat protein fused to the DNA-binding protein LexA; the RNA-binding domain to be analyzed is fused to the transcriptional activation domain of the yeast Gal4 protein; and the bifunctional RNA consists of binding sites for the coat protein and for the other RNA-binding domain. We built an RNA library such that short fragments of genomic DNA from yeast were transcribed in yeast together with binding sites for the coat protein. We screened this hybrid RNA library for RNAs that bound to the yeast Snp1 protein, a homolog of the human U1-70K protein. The screen yielded as the strongest positive the fragment of U1 RNA that contains loop I, which is known to bind to Snp1 in U1 snRNP. We also identified four other RNA ligands that produced weaker three-hybrid signals, suggesting lower affinities for Snp1 as compared to U1 RNA. In addition, this search also yielded a set of RNA sequences that can activate transcription on their own when bound to a promoter through a protein interaction.

5] Yeast three-hybrid system to detect and analyze interactions between RNA and protein

Publisher Summary This article describes the use of a different genetic approach to detect RNA protein-interactions. In this method, termed the three-hybrid system, the binding of a hybrid RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo . The method can be used to detect and analyze RNA-protein interaction when both partners are known or to find a mate when only one is known. One asset of the three-hybrid method, shared with other genetic approaches to finding protein partners, is that a DNA clone of the interacting protein is obtained. With the proliferation of sequence data bases and genomic information, a small bit of sequence information may be enough to help determine whether that protein is a legitimate partner or shed light on its function.

Use of the Yeast Three-Hybrid System as a Tool to Study Caspases

Caspases are a family of heteromeric (p20/p10) cysteine proteases with important functions in the regulation of apoptosis and inflammation. Up to now, tools to identify new substrates for caspases have mostly been limited to the random screening ofin vitrotranslated proteins that are known, or assumed, to play a role in apoptosis. We describe the use of a yeast three-hybrid approach as a tool that adapts the classical two-hybrid system to the needs of heteromeric caspases for functional dissection of known interactions or screening for physiological substrates and inhibitors. Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that were each fused to the Gal4 DNA-binding domain. Upon coexpression of a third hybrid of the Gal4 activation domain and the viral caspase-1 pseudosubstrate inhibitors CrmA or p35, or the prototype physiological caspase-1 substrate prointerleukin-1β, a functional Gal4 transcription factor could be reconstituted. In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of caspase-1. Therefore, the three-hybrid system might allow screening for new physiological substrates and inhibitors of heteromeric caspases.

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system.

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.

Technological advances in high-throughput screening

A variety of assay technologies continue to be developed for high-throughput screening. These include cell-based assays, surrogate systems using microbial cells such as yeast and bacterial two-hybrid and three-hybrid systems, and systems to measure nucleic acid-protein and receptor-ligand interactions. Modifications have been developed for cell-free, homogeneous assay systems, such as time-resolved fluorescence, fluorescence polarization and the scintillation proximity assay. Innovations in engineering and chemistry have led to delivery systems for nanoliter volumes and sensitive biosensors for ultra-high-throughout screening conducted in nanoliter and picoliter volumes. Spectroscopic methods have been extended to read single molecule fluorescence. Technologies are being developed to identify new targets from genomic information in order to design the next generation of screens.

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.

A three-hybrid system for detecting small ligand-protein receptor interactions.

Small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo. This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined. The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506. Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone-FK506 heterodimer. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor-LexA DNA binding domain fusion protein in the presence of dexamethasone-FK506 heterodimer. We isolated overlapping clones of human FKBP12. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.

A Yeast Three-Hybrid Method to Clone Ternary Protein Complex Components

The yeast two-hybrid system is a powerful technique to detect binary protein interactions. In order to facilitate ternary complex analysis, we have developed the yeast three-hybrid system. Using epidermal growth factor (EGF) receptor, Grb2 and Sos proteins as an example, we demonstrated the use of the three-hybrid system to detect formation of a three-component complex, i.e., the Grb2-mediated interactions between EGF receptor and Sos. We then used the three-hybrid system to screen a library with Gal4–DNA binding domain –EGF receptor and Gal4 activation domain–Sos fusion proteins and isolated a plasmid encoding Grb2. Thus, the three-hybrid system is useful for characterizing and cloning components of ternary protein complexes.