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

König, Julian; Julius, Christian; Baumann, Sebastian; Homann, Matthias; Göringer, H. Ulrich; Feldbrügge, Michael

doi:10.1261/rna.334307

Cited by 27 publications

(31 citation statements)

References 35 publications

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“…A protein (''prey'') is expressed as a fusion with the Gal4 transcription activation domain. The Y3H has been successfully used to confirm natural or artificial RNAprotein interactions and to identify novel RNA partners for target proteins by selection from RNA or protein libraries (SenGupta et al 1996;Zhang et al 1999Zhang et al , 2000Kraemer et al 2000;Maher 2001, 2003;van Zon et al 2001;Bernstein et al 2002Bernstein et al , 2005Sokolowski et al 2003;Spiridonov et al 2003;Klimek-Tomczak et al 2004;Hook et al 2005;Riley et al 2006;Seay et al 2006;Konig et al 2007;Stumpf et al 2008;Koh et al 2009;Wurster et al 2009). …”

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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“…Gal /min*RFU Gfp ; König et al 2007). lacZ reporter gene expression was scored by measuring b-galactosidase activity using a fluorogenic substrate.…”

Section: Drfumentioning

confidence: 99%

“…As a first step to answer these questions, we applied the reverse yeast three-hybrid system. This powerful technique enables investigation of interactions between RNA and proteins in vivo (SenGupta et al 1996(SenGupta et al , 1999Stumpf et al 2008) as well as mapping of the binding domain at ease (König et al 2007). We discovered that the sequence AUACCC is necessary and sufficient for binding by Khd4.…”

Section: Khd4 Recognizes Auaccc In Vivo Via Tandem Kh Domains 3 Andmentioning

confidence: 99%

“…Plasmids pCR2.1-and pCRII-Topo for cloning (Invitrogen), and pACTIIA as well as pIIIMS2-1 for the yeast three-hybrid analysis were used (SenGupta et al 1996;König et al 2007). Plasmid constructions are described in the Supplemental Materials and Methods.…”

Section: Plasmids and Plasmid Constructionsmentioning

confidence: 99%

“…These were size-selected for 50-250 bp using agarose gel electrophoresis followed by electroelution and ligation with vector pIIIMS2-4 (König et al 2007), which was linearized with ClaI and dephosphorylated. Upon transformation in E. coli, plasmid DNA was extracted from a total of 1.5 3 10 6 transformants to obtain the library.…”

Section: Reverse Yeast Three-hybrid Systemmentioning

confidence: 99%

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Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity inUstilago maydis

Vollmeister¹,

Haag²,

Zarnack³

et al. 2009

RNA

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RNA-binding proteins constitute key factors of the post-transcriptional machinery. These regulatory proteins recognize specific elements within target transcripts to promote, for example, maturation, translation, or stability of mRNAs. In Ustilago maydis, evidence is accumulating that post-transcriptional processes are important to determine pathogenicity. Deletion of khd4, encoding a predicted RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence. Here, we demonstrate that Khd4 recognizes the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence most likely functions as a regulatory RNA element in U. maydis, since it accumulates in 39 untranslated regions. Consistently, an independent mRNA expression profiling approach revealed that the binding motif is significantly enriched in transcripts showing altered expression levels in khd4D strains. Since the vast majority of potential Khd4 target mRNAs exhibit increased amounts in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants, which exhibit altered cell morphology, disturbed filamentous growth, and severely reduced virulence. Hence, RNA binding is essential for function of Khd4, stressing the importance of post-transcriptional control in regulating morphology and pathogenicity.

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Two‐ and Three‐Hybrid Systems

Gestwicki

Kumar

2008

Wiley Encyclopedia of Chemical Biology

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One goal of chemical biology is to document and understand the macromolecular interactions that take place in the cell. In this review, we discuss the classic yeast two‐hybrid method (and its derivatives) and how this method continues to provide insight into the number of protein–protein partnerships in a cell. In turn, these platforms have been adapted to explore a variety of interactions, such as those between proteins and small molecules. These systems collectively are called three‐hybrid assays, and they provide new opportunities for the discovery of potential binding pairs. However, two‐ and three‐hybrid assays also have significant disadvantages, such as incomplete coverage and high rates of false positives. With these issues in mind, we discuss emerging ways of minimizing the impact of these limitations using microarrays and mass spectrometry. Finally, chemical probes related to the three‐hybrid concept are going beyond observation and providing active, rational control over individual protein–protein contacts. In these systems, bifunctional compounds are used to homo‐ or heterodimerize target proteins reversibly, thus altering their colocalization. By purposefully controlling protein–protein contacts, these chemical dimerization methods have progressed beyond observation and towards synthetic manipulation of protein function.

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

Cited by 27 publications

References 35 publications

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

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

Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity inUstilago maydis

Two‐ and Three‐Hybrid Systems

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