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

SenGupta, Dhruba J.; Wickens, Marvin; Fields, Stanley

doi:10.1017/s1355838299002113

Cited by 63 publications

(61 citation statements)

References 13 publications

(6 reference statements)

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“…1), an ingenious method to assay RNA-protein interactions in vivo. Identifying RNAprotein interactions involves transformation of a yeast strain with RNA and protein expression plasmids, followed by assays of HIS3 and lacZ reporter genes (SenGupta et al 1996(SenGupta et al , 1999). An experimental RNA insert (''bait'') is cloned into an RNA expression vector, for example, pIIIA-MS2-2, which provides the molecular recognition required for tethering to yeast chromatin upstream of HIS3 and lacZ reporter genes (Fig.…”

Section: Introductionmentioning

confidence: 99%

Selections that optimize RNA display in the yeast three-hybrid system

Wurster

Maher²

2009

RNA

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The yeast three-hybrid system (Y3H) is a powerful tool to select or confirm RNA-protein interactions. Target protein recognition of an RNA insert within a test transcript depends on at least three factors: intrinsic protein affinity for the properly folded insert, retention of RNA insert tertiary structure within a longer RNA transcript, and accessibility of the RNA insert to the target protein. Y3H reporter gene readout reflects the combination of these factors. Here, we discuss RNA insert tertiary structure and accessibility in the Y3H as ''RNA display.'' We review evidence that RNA display can sometimes be optimized during Y3H selections that do not increase the intrinsic affinity of an RNA insert for a target protein. This situation is more likely when a library of RNA inserts and heterogeneous flanking sequences is subjected to selection, and is less likely when point mutations are targeted to the insert in a fixed context. An RNA display vector with enhanced modularity has been developed to minimize sequence context effects in the Y3H.

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Section: Introductionmentioning

confidence: 99%

Selections that optimize RNA display in the yeast three-hybrid system

Wurster

Maher²

2009

RNA

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“…The yeast three-hybrid system is a powerful technique to investigate interactions between RNA and proteins in vivo (SenGupta et al 1996(SenGupta et al , 1999Hook et al 2005). It allows the identification of RNA and protein binding partners as well as the dissection of higher-order RNA/protein complexes (Bernstein et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

König

Julius

Baumann

et al. 2007

RNA

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Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motifcontaining RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.

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“…The yeast three-hybrid system is a sensitive in vivo assay for detecting RNA-protein interactions ( Fig. 1A; SenGupta et al 1996SenGupta et al , 1999Zhang et al 1999;Kraemer et al 2000;Zhang et al 2000;Hook et al 2005). Transcription of HIS3 and LacZ reporter genes depends on a trimolecular interaction within the yeast nucleus (SenGupta et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system

Riley

Cassiday

Kumar

et al. 2006

RNA

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The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -nonspecific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast threehybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.

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

Cited by 63 publications

References 13 publications

Selections that optimize RNA display in the yeast three-hybrid system

Selections that optimize RNA display in the yeast three-hybrid system

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system

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